Method for producing an organosilicon product

ABSTRACT

The present invention provides compositions and methods for catalyzing the formation of carbon-silicon bonds using heme proteins. In certain aspects, the present invention provides heme proteins, including variants and fragments thereof, that are capable of carrying out in vitro and in vivo carbene insertion reactions for the formation of carbon-silicon bonds. In other aspects, the present invention provides methods for producing an organosilicon product, the method comprising providing a silicon-containing reagent, a carbene precursor, and a heme protein; and combining the components under conditions sufficient to produce an organosilicon product. Host cells expressing the heme proteins are also provided by the present invention.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/422,360 filed on Feb. 1, 2017, which claims benefit of U.S.Provisional Application No. 62/290,211 filed on Feb. 2, 2016; U.S.Provisional Application No. 62/365,797 filed on Jul. 22, 2016; and U.S.Provisional Application No. 62/409,137 filed on Oct. 17, 2016, thedisclosures of which are incorporated herein by reference in theirentirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No.CBET1403077 awarded by the National Science Foundation and Grant No.GM007616 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

The Sequence Listing written in fileSequenceListing_086544-020330US-1025419.txt created on Mar. 10, 2017,3,274 bytes, machine format IBM-PC, MS-Windows operating system, ishereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Organic compounds containing carbon-silicon bonds are of great interestin the fields of synthetic chemistry, drug discovery, nuclear medicine,biotechnology, and materials science. As a result, chemical methodsavailable for introducing silicon to the carbon framework of organicmolecules have improved in recent years. Among these methods, however,only a small fraction are suitable for the preparation of chiralorganosilicon compounds. One approach for asymmetric carbon-silicon bondformation is via carbenoid insertion into silicon-hydrogen bonds, whichcan be achieved using chiral rhodium, iridium, or copper catalysts.While these transition metal catalysts have demonstrated utility inpreparing highly selective products, their turnovers are poor (i.e.,none exceeds a total turnover number of 100), and the elaborate catalyststructures required to control selectivity are lengthy and expensive tosynthesize. Moreover, most of these processes rely on the use ofchlorinated solvents and cryogenic conditions to achieve the desiredreaction outcomes. Accordingly, there is a need in the art for newmethods of synthesis of organosilicon compounds, in particular methodsthat can carry out the production of these compounds with highefficiency, low cost, and reduced dependence on harsh chemical reagentsand reaction conditions. The present invention satisfies this need, andprovides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides a cytochrome c proteinvariant, or a fragment thereof, that is capable of enantioselectivelycatalyzing the formation of a carbon-silicon bond. In some embodiments,the cytochrome c protein variant comprises a mutation at an axial hemecoordination residue. In other embodiments, the cytochrome c proteinvariant comprises one or more mutations selected from the groupconsisting of V75, M100, and M103 relative to the amino acid sequenceset forth in SEQ ID NO:1. In some instances, the one or more mutationscomprise a V75 mutation and an M100 mutation relative to the amino acidsequence set forth in SEQ ID NO:1. In other instances, the one or moremutations comprise a M100 mutation and an M103 mutation relative to theamino acid sequence set forth in SEQ ID NO:1. In yet other instances,the one or more mutations comprise a V75 mutation, a M100 mutation, andan M103 mutation relative to the amino acid sequence set forth in SEQ IDNO:1. In particular instances, the V75 mutation is a V75T mutation. Inother instances, the M100 mutation is an M100D or M100E mutation. Instill other instances, the M103 mutation is an M103E mutation.

In other embodiments, the heme cofactor of the cytochrome c proteinvariant is a non-native cofactor.

In some embodiments, the cytochrome c protein variant has a higher totalturnover number (TTN) compared to the wild-type protein. In someinstances, the TTN is greater than about 70. In particular instances,the TTN is greater than about 1,800.

In other embodiments, the cytochrome c protein variant has a higherturnover frequency (TOF) compared to the wild-type protein. In someinstances, the TOF is at least about 2-fold greater than the wild-typeprotein. In particular instances, the TOF is at least about 7-foldgreater than the wild-type protein. In some instances, the TOF is atleast about 10 min⁻¹. In particular instances, the TOF is at least about45 min⁻¹.

In some embodiments, the cytochrome c protein variant produces anorganosilicon product with a % ee of at least about 75%. In someinstances, the cytochrome c protein variant produces an organosiliconproduct with a % ee of at least about 95%. In particular instances, thecytochrome c protein variant produces an organosilicon product with a %ee of at least about 99%.

In a second aspect, the invention provides a cell comprising acytochrome c protein variant, or a fragment thereof, of the presentinvention. In some embodiments, the cell is a bacterial, archaeal,yeast, or fungal cell. In some instances, the bacterial cell is anEscherichia coli cell.

In a third aspect, the invention provides a method for producing anorganosilicon product. In some embodiments, the method comprisescombining a silicon-containing reagent, a carbene precursor, and a hemeprotein, a fragment thereof, or a variant thereof, under conditionssufficient to produce an organosilicon product. In particularembodiments, the heme protein or fragment thereof is selected from thegroup consisting of a cytochrome protein, a globin protein, a myoglobinprotein, a hemoglobin protein, a peroxidase, a catalase, and acombination thereof. In some instances, the globin protein is fromMethylacidiphilum infernorum. In other instances, the cytochrome proteinis a cytochrome c protein, a cytochrome P450 protein, or a combinationthereof. In some instances, the cytochrome P450 protein is a cytochromeP450 BM3 (CYP102A1) protein. In particular instances, the cytochrome cprotein is selected from the group consisting of Rhodothermus marinus(Rma) cytochrome c, Rhodopila globiformis cytochrome c, Hydrogenobacterthermophilus cytochrome c, Saccharomyces cerevisiae cytochrome c, horseheart cytochrome c, bovine heart cytochrome c, and a combinationthereof.

In some embodiments, the heme protein, fragment thereof, or variantthereof can enantioselectively catalyze the formation of acarbon-silicon bond. In other embodiments, the heme protein variantcomprises a mutation at an axial heme coordination residue. In someembodiments, the heme variant is a Rhodothermus marinus (Rma) cytochromec protein variant comprising one or more mutations selected from thegroup consisting of V75, M100, and M103 relative to the amino acidsequence set forth in SEQ ID NO:1. In some instances, the one or moremutations comprise a V75 mutation and an M100 mutation relative to theamino acid sequence set forth in SEQ ID NO:1. In other instances, theone or more mutations comprise an M100 mutation and an M103 mutationrelative to the amino acid sequence set forth in SEQ ID NO:1. Inparticular instances, the one or more mutations comprise a V75 mutation,an M100 mutation, and an M103 mutation relative to the amino acidsequence set forth in SEQ ID NO:1. In some instances, the V75 mutationis a V75T mutation. In other instances, the M100 mutation is an M100D orM100E mutation. In still other instances, the M103 mutation is an M103Emutation.

In other embodiments, the heme protein cofactor is a non-nativecofactor.

In some other embodiments, the heme protein is a variant that has ahigher TTN compared to the wild-type protein. In some instances, the TTNis greater than about 70. In particular instances, the TTN is greaterthan about 1,800.

In other embodiments, the heme protein has a TOF that is at least about10 min⁻¹. In some instances, the TOF is at least about 45 min⁻¹. In someembodiments, the heme protein is a variant that has a TOF that is highercompared to the TOF of the wild-type protein. In some instances, the TOFis at least about 2-fold greater than the wild-type protein. Inparticular instances, the TOF is at least about 7-fold greater than thewild-type protein.

In still other embodiments, the heme protein, fragment thereof, orvariant thereof produces an organosilicon product with a % ee of atleast about 75%. In some instances, the % ee is at least about 95%. Inparticular instances, the % ee is at least about 99%.

In some embodiments, the silicon-containing reagent is a compoundaccording to Formula I:

wherein R¹, R², R³ and R⁴ are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl,C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted6- to 10-membered heteroaryl, optionally substituted 6- to 10-memberedheterocyclyl, cyano, halo, hydroxy, alkoxy, SR⁷, N(R⁸)₂, B(R⁹)₂,Si(R⁹)₃, P(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂ andC(O)NR⁷OR⁸; and wherein R⁷, R⁸, and R⁹ are independently selected fromthe group consisting of H, optionally substituted C₁₋₁₈ alkyl, C₂₋₁₈alkenyl, C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionallysubstituted 6- to 10-membered heteroaryl, and optionally substituted 6-to 10-membered heterocyclyl.

In some instances, R² is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl. In other instances, R³ and R⁴ are C₁₋₆ alkyl.

In other embodiments, the carbene precursor is a diazo substrateaccording to Formula II:

wherein R⁵ and R⁶ are independently selected from the group consistingof H, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈haloalkyl, optionally substituted C₂₋₁₈ alkenyl, optionally substitutedC₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted6- to 10-membered heteroaryl, optionally substituted 6- to 10-memberedheterocyclyl, cyano, halo, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷,C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷, andP(O)(OR⁷)₂; and wherein each R⁷, R⁸, and R⁹ is independently selectedfrom the group consisting of H and optionally substituted C₁₋₆ alkyl.

In some instances, R⁵ is C(O)OR⁷. In other instances, R⁶ is selectedfrom the group consisting of optionally substituted C₁₋₁₈ alkyl,optionally substituted C₁₋₁₈ haloalkyl, and optionally substituted C₂₋₁₈alkenyl.

In some embodiments, the organosilicon product is a compound accordingto Formula III:

wherein R¹, R², R³ and R⁴ are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl,C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted6- to 10-membered heteroaryl, optionally substituted 6- to 10-memberedheterocyclyl, cyano, halo, hydroxy, alkoxy, SR⁷, N(R⁸)₂, B(R⁹)₂,Si(R⁹)₃, P(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂ andC(O)NR⁷OR⁸. R⁵ and R⁶ are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl,C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted6- to 10-membered heteroaryl, optionally substituted 6- to 10-memberedheterocyclyl, cyano, halo, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷,C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷ and P(O)(OR7)₂.R⁷, R⁸, and R⁹ are independently selected from the group consisting ofH, optionally substituted C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl.

In some instances, R² is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl. In other instances, R³ and R⁴ are C₁₋₆ alkyl. In yet otherinstances, R⁵ is C(O)OR⁷. In other instances, R⁶ is selected from thegroup consisting of optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₁₋₁₈ haloalkyl, and optionally substituted C₂₋₁₈ alkenyl.

In other embodiments, a reducing agent is combined with thesilicon-containing reagent and carbene precursor. In some embodiments,the organosilicon product is produced in vitro. In still otherembodiments, the heme protein, fragment thereof, or variant thereof isexpressed in a cell and the organosilicon product is produced in vivo.In some instances, the cell is a bacterial cell, archaeal cell, yeastcell, or fungal cell. In particular instances, the bacterial cell is anEscherichia coli cell. In some embodiments, the organosilicon product isproduced under anaerobic conditions. In other embodiments, theorganosilicon product is produced under aerobic conditions.

In a fourth aspect, the invention provides a reaction mixture forproducing an organosilicon product. In some embodiments, the reactionmixture comprises a silicon-containing reagent, a carbene precursor, anda heme protein, a fragment thereof, or a variant thereof. In particularembodiments, the heme protein or fragment thereof is selected from thegroup consisting of a cytochrome protein, a globin protein, a myoglobinprotein, a hemoglobin protein, a peroxidase, a catalase, and acombination thereof. In some instances, the globin protein is selectedfrom the group consisting of Methylacidiphilum infernorum globinprotein, sperm whale globin protein, Rhodothermus marinus (Rma) globinprotein, Bacillus subtilis globin protein, Pyrobaculum ferrireducensglobin protein, Aeropyrum pernix globin protein, Campylobacter jejuniglobin protein, and a combination thereof. In other instances, thecytochrome protein is a cytochrome c protein, a cytochrome P450 protein,or a combination thereof. In some instances, the cytochrome P450 proteinis a cytochrome P450 BM3 (CYP102A1) protein. In particular instances,the cytochrome c protein is selected from the group consisting ofRhodothermus marinus (Rma) cytochrome c, Rhodopila globiformiscytochrome c, Hydrogenobacter thermophilus cytochrome c, Saccharomycescerevisiae cytochrome c, horse heart cytochrome c, bovine heartcytochrome c, and a combination thereof.

In some embodiments, the heme protein, fragment thereof, or variantthereof can enantioselectively catalyze the formation of acarbon-silicon bond. In other embodiments, the heme protein variantcomprises a mutation at an axial heme coordination residue. In someembodiments, the heme variant is a Rhodothermus marinus (Rma) cytochromec protein variant comprising one or more mutations selected from thegroup consisting of V75, M100, and M103 relative to the amino acidsequence set forth in SEQ ID NO:1. In some instances, the one or moremutations comprise a V75 mutation and an M100 mutation relative to theamino acid sequence set forth in SEQ ID NO:1. In other instances, theone or more mutations comprise an M100 mutation and an M103 mutationrelative to the amino acid sequence set forth in SEQ ID NO:1. Inparticular instances, the one or more mutations comprise a V75 mutation,an M100 mutation, and an M103 mutation relative to the amino acidsequence set forth in SEQ ID NO:1. In some instances, the V75 mutationis a V75T mutation. In other instances, the M100 mutation is an M100D orM100E mutation. In still other instances, the M103 mutation is an M103Emutation.

In other embodiments, the heme protein cofactor is a non-nativecofactor.

In some other embodiments, the heme protein is a variant that has ahigher TTN compared to the wild-type protein. In some instances, the TTNis greater than about 70. In particular instances, the TTN is greaterthan about 1,800.

In other embodiments, the heme protein has a TOF that is at least about10 min⁻¹. In some instances, the TOF is at least about 45 min⁻¹. In someembodiments, the heme protein is a variant that has a TOF that is highercompared to the TOF of the wild-type protein. In some instances, the TOFis at least about 2-fold greater than the wild-type protein. Inparticular instances, the TOF is at least about 7-fold greater than thewild-type protein.

In still other embodiments, the heme protein, fragment thereof, orvariant thereof produces an organosilicon product with a % ee of atleast about 75%. In some instances, the % ee is at least about 95%. Inparticular instances, the % ee is at least about 99%.

In some embodiments, the silicon-containing reagent is a compoundaccording to Formula I:

wherein R¹ is H and R², R³, and R⁴ are independently selected from thegroup consisting of H, optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted C₆₋₁₀aryl-C₁₋₆ alkyl, optionally substituted 6- to 10-membered heteroaryl,and optionally substituted 6- to 10-membered heterocyclyl; provided thatat least one of R², R³, and R⁴ is other than H.

In some instances, R² is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl. In other instances, R³ and R⁴ are C₁₋₆ alkyl.

In other embodiments, the carbene precursor is a diazo substrateaccording to Formula II:

wherein R⁵ and R⁶ are independently selected from the group consistingof H, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈haloalkyl, optionally substituted C₂₋₁₈ alkenyl, optionally substitutedC₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted6- to 10-membered heteroaryl, optionally substituted 6- to 10-memberedheterocyclyl, cyano, halo, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷,C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷, andP(O)(OR⁷)₂; and wherein each R⁷, R⁸, and R⁹ is independently selectedfrom the group consisting of H and optionally substituted C₁₋₆ alkyl.

In some instances, R⁵ is C(O)OR⁷. In other instances, R⁶ is selectedfrom the group consisting of optionally substituted C₁₋₁₈ alkyl,optionally substituted C₁₋₁₈ haloalkyl, and optionally substituted C₂₋₁₈alkenyl.

In some embodiments, the organosilicon product is a compound accordingto Formula III:

wherein R¹ is H; R², R³, and R⁴ are independently selected from thegroup consisting of H, optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted C₆₋₁₀aryl-C₁₋₆ alkyl, optionally substituted 6- to 10-membered heteroaryl,and optionally substituted 6- to 10-membered heterocyclyl; provided thatat least one of R², R³, and R⁴ is other than H; and R⁵ and R⁶ areindependently selected from the group consisting of H, optionallysubstituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈ haloalkyl,optionally substituted C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to10-membered heteroaryl, optionally substituted 6- to 10-memberedheterocyclyl, cyano, halo, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷,C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷, andP(O)(OR⁷)₂; wherein each R⁷, R⁸, and R⁹ is independently selected fromthe group consisting of H and optionally substituted C₁₋₆ alkyl.

In some instances, R² is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl. In other instances, R³ and R⁴ are C₁₋₆ alkyl. In yet otherinstances, R⁵ is C(O)OR⁷. In other instances, R⁶ is selected from thegroup consisting of optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₁₋₁₈ haloalkyl, and optionally substituted C₂₋₁₈ alkenyl.

In other embodiments, the reaction mixture further comprises a reducingagent. In some embodiments, the organosilicon product is produced invitro. In still other embodiments, the heme protein, fragment thereof,or variant thereof is expressed in a cell and the organosilicon productis produced in vivo. In some instances, the cell is a bacterial cell,archaeal cell, yeast cell, or fungal cell. In particular instances, thebacterial cell is an Escherichia coli cell. In some embodiments, theorganosilicon product is produced under anaerobic conditions. In otherembodiments, the organosilicon product is produced under aerobicconditions.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative yield and % ee of organosilicon productsafforded by various heme proteins. Myoglobin protein tested: Mb H64VV68A. Cytochrome P450 proteins tested: WT T268A, T268A AxS, T268A AxY,T268A AxH, T268A AxA, F87A C400S T268A, C3C T438S, C3C C400S T438S, A1003/06, P-I263F Heme, H2-5-F10, Hstar H92N H100N, Hstar H400S, and P411CIS T438S. Globin tested: HGG Y29V Q50A. Cytochrome c proteins tested:Hth WT, Rgl WT and Rma WT.

FIG. 2 shows total turnover number (TTN) and % ee of various Rma cyt cvariants in catalysis of production of organosilicon compounds.

FIG. 3 shows % ee of various Rma cyt c variants in whole-cell catalysisof production of organosilicon compounds.

FIG. 4 shows the chemical structures of some known catalytic systems.Labels correspond to those used in Table 2.

FIGS. 5A-5E show heme protein-catalyzed carbon-silicon bond formation.FIG. 5A shows carbon—silicon bond formation catalyzed by heme andpurified heme proteins. Various P450s and myoglobin also catalyzed theformation of carbon—silicon bonds, but the reactions were notenantioselective. FIG. 5B shows a surface representation of theheme-binding pocket of wild-type Rma cyt c (PDB ID: 3CP5). FIG. 5C showsthe “active site” structure of wild-type Rma cyt c showing a covalentlybound heme cofactor ligated by axial ligands H49 and M100. Amino acidresidues M100, V75 and M103 residing close to the heme iron weresubjected to site-saturation mutagenesis. FIG. 5D shows the results ofdirected evolution of Rma cyt c for carbon—silicon bond formation(reaction shown in FIG. 5A). Experiments were performed using lysates ofE. coli expressing Rma cyt c variant (OD₆₀₀=15; heat-treated at 75° C.for 10 minutes), 10 mM silane, 10 mM diazo ester, 10 mM Na₂S₂O₄, 5 vol %MeCN, M9-N buffer (pH 7.4) at room temperature under anaerobicconditions for 1.5 h. Reactions were performed in triplicate. FIG. 5Eshows carbon—silicon bond forming rates over four generations of Rma cytc.

FIG. 6 shows a graph of product formation as a function of time for fourgenerations of Rma cyt c.

FIG. 7 shows the scope of Rma cyt c V75T M100D M103E-catalyzedcarbon—silicon bond formation and the chemical structures oforganosilicon products Compounds 3-22. Standard reaction conditionswere: lysate of E. coli expressing Rma cyt c V75T M100D M103E(OD₆₀₀=1.5; heat-treated at 75° C. for 10 minutes), 20 mM silane, 10 mMdiazo ester, 10 mM Na₂S₂O₄, 5 vol % MeCN, M9-N buffer (pH 7.4) at roomtemperature under anaerobic conditions. Reactions were performed intriplicate. [a] OD₆₀₀=5 lysate. [b] OD₆₀₀=0.5 lysate. [c] OD₆₀₀=15lysate. [d] 10 mM silane. [e] OD₆₀₀=0.15 lysate.

FIGS. 8A and 8B show the chemoselectivity and in vivo activity ofevolved Rma cyt c. FIG. 8A shows that chemoselectivity for carbene Si—Hinsertion over N—H insertion increased dramatically during the directedevolution of Rma cyt c. Reaction conditions as described in FIG. 7 wereused. Reactions were performed in duplicate using heat-treated lysatesof E. coli expressing Rma cyt c with protein concentration normalizedacross variants. Product distribution was quantified after 2 hours ofreaction time (i.e., before complete conversion, no double insertionproduct was observed under these conditions). FIG. 8B shows the in vivosynthesis of organosilicon compound 22.

FIGS. 9A and 9B show ferrous assay calibration curves. FIG. 9A shows thecalibration curve for wild-type Rma cyt c. FIG. 9B shows the calibrationcurve for Rma cyt c V75T M100D M103E.

FIG. 10 shows a preparative-scale whole-cell biocatalytic reaction inwhich Compound 22 was synthesized using Compound 23 as thesilicon-containing reagent and Me-EDA as the diazo substrate.

FIG. 11 shows a representative SDS-PAGE gel of purified wild-type Rmacyt c and its V75T M100D M103E variant (TDE) in comparison to a standardprotein ladder. The second and third lanes from the left are the samesamples loaded at lower protein concentrations.

FIG. 12 shows circular dichroism (CD) spectra of purified Rma cyt c V75TM100D M103E (Rma TDE). Rma cyt c V75T M100D M103E was purified withoutperforming the heat treatment step. Two identical samples were preparedin M9-N buffer, and one was heat treated at 75° C. for 10 minutes. Aftercooling to room temperature, the samples were analyzed by CD. The CDspectra of heat-treated and untreated Rma cyt c V75T M100D M103E wereidentical, suggesting that heat treatment at 75° C. for 10 minutes didnot cause irreversible denaturation of the protein. The Δϵ_(MRW) valuesshown are similar to previously published values for wild-type Rma cyt c(Stelter et al. Biochemistry 47:11953-11963 (2008)), which shows thatmutations V75T, M100D, and M103E were not highly disruptive to theprotein secondary structure.

FIGS. 13A-13C show a binding mode for the iron carbenoid in wild-typeRma cyt c and reaction trajectory for carbon—silicon bond formation.FIG. 13A shows a reaction in which a carbon-silicon bond is formed. FIG.13B shows the active site structure of wild-type Rma cyt c showingresidues V75, M100, and M103 (PDB ID: 3CP5). FIG. 13C shows the bindingmode for the iron-carbenoid, where the carbenoid forms in a way thattakes the place of the axial methionine. The silane approaches from themore solvent-exposed side in the wild-type protein, which explains theobserved stereochemistry of the organosilicon product. The V75T, M100D,and M103E mutations may promote reactivity by improving solvent andsubstrate access to the iron center.

FIGS. 14A and 14B show an example of a reaction time course of apurified Rma cyt c M100D V75T M103E-catalyzed reaction betweenphenyldimethylsilane and Me-EDA. FIG. 14A shows a schematic of thereaction. FIG. 14B shows TTN as a function of time. Experiments wereperformed using 3 μM purified Rma cyt c V75T M100D M103E, 10 mM silane,10 mM diazo ester, 10 mM Na₂S₂O₄, 5 vol % MeCN, M9-N buffer at roomtemperature under anaerobic conditions. Reactions were performed induplicate. TTNs shown are the average of two experiments.

FIG. 15 shows additional substrates that were tested for Rma cyt c V75TM100D M103E-catalyzed carbon—silicon bond formation. Rma cyt c V75TM100D M103E-catalyzed reactions of silanes (Compounds Si—A—H) weretested with Me-EDA and that of diazo compounds (Compounds diazo-A-C)were tested with phenyldimethylsilane under standard in vitrobiocatalytic reaction conditions. Product formation was analyzed byGC-MS. For

Compounds Si—A—D and diazo-A, formation of organosilicon product wasdetected by GC-MS. For Compounds Si—E—H and diazo-B-C, formation oforganosilicon product was not detected by GC-MS.

FIGS. 16A and 16B show Hammett analysis of Rma cyt c V75T M100DM103E-catalyzed carbon—silicon bond formation. FIG. 16A shows thechemical reaction. FIG. 16B shows a Hammett plot that shows a smallbuild-up of positive charge on the silane in the reaction transitionstate. This observation is similar to that reported for carbeneinsertion into Si—H bond catalyzed by copper (ρ=−0.54) (Dakin et al,Organometallics 19:2896-2908 (2000)) and rhodium (ρ=−0.31) (Landais etal. Tetrahedron Lett. 38:229-232 (1997)).

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Enzymes are environmentally friendly and cost-effective alternatives totransition metal catalysts. These genetically encoded catalysts canachieve exquisite selectivity with efficiency that is difficult orimpossible to achieve using small molecule catalysts. In nature,however, biologically synthesized carbon-silicon bonds, either in vitroor in vivo, are not known, despite silicon being the second mostabundant element in the Earth's crust.

The present invention is based, in part, on the discovery that variousheme proteins catalyze the in vitro and in vivo formation ofcarbon-silicon bonds. In addition, the present invention is based, inpart, on the use of directed protein evolution to improve the ability ofheme proteins to construct carbon-silicon chemical bonds. As such, thepresent invention provides, among other things, engineered heme proteinsthat catalyze carbon-silicon bond formation with a high total turnovernumber and high enantioselectivity.

II. Definitions

Unless specifically indicated otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this invention belongs. Inaddition, any method or material similar or equivalent to a method ormaterial described herein can be used in the practice of the presentinvention. For purposes of the present invention, the following termsare defined.

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the reagent” includes reference to one or more reagentsknown to those skilled in the art, and so forth.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typical, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

The terms “heme protein variant” and “heme enzyme variant” include anyheme-containing enzyme comprising at least one amino acid mutation withrespect to wild-type and also include any chimeric protein comprisingrecombined sequences or blocks of amino acids from two, three, or moredifferent heme-containing enzymes.

The term “whole cell catalyst” includes cells expressing heme-containingenzymes, wherein the whole cell catalyst displays carbon-silicon bondformation activity.

The term “carbene precursor” includes molecules that can be decomposedin the presence of metal (or enzyme) catalysts to structures thatcontain at least one divalent carbon with only 6 valence shell electronsand that can be transferred to a silicon-hydrogen bond, a silicon-carbonbond, a silicon-sulfur bond, a silicon-nitrogen bond, a silicon-boronbond, a silicon-silicon bond, or a silicon-phosphorus bond to formvarious carbon ligated products. Examples of carbene precursors include,but are not limited to, diazo reagents, diazirene reagents, and epoxidereagents.

As used herein, the term “anaerobic”, when used in reference to areaction, culture or growth condition, is intended to mean that theconcentration of oxygen is less than about 25 μM, preferably less thanabout 5 μM, and even more preferably less than 1 μM. The term is alsointended to include sealed chambers of liquid or solid medium maintainedwith an atmosphere of less than about 1% oxygen. Preferably, anaerobicconditions are achieved by sparging a reaction mixture with an inert gassuch as nitrogen or argon.

As used herein, the term “alkyl” refers to a straight or branched,saturated, aliphatic radical having the number of carbon atomsindicated. Alkyl can include any number of carbons, such as C₁₋₂, C₁₋₃,C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆,C₄₋₅, C₄₋₆ and C₅₋₆. For example, C₁₋₆ alkyl includes, but is notlimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can refer toalkyl groups having up to 20 carbons atoms, such as, but not limited toheptyl, octyl, nonyl, decyl, etc. Alkyl groups can be optionallysubstituted with one or more moieties selected from halo, hydroxy,amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, andcyano.

As used herein, the term “alkenyl” refers to a straight chain orbranched hydrocarbon having at least 2 carbon atoms and at least onedouble bond. Alkenyl can include any number of carbons, such as C₂,C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆,C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆. Alkenyl groups can have any suitablenumber of double bonds, including, but not limited to, 1, 2, 3, 4, 5 ormore. Examples of alkenyl groups include, but are not limited to, vinyl(ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl,butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl,1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl,1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl.Alkenyl groups can be optionally substituted with one or more moietiesselected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl,carboxy, amido, nitro, oxo, and cyano.

As used herein, the term “alkynyl” refers to either a straight chain orbranched hydrocarbon having at least 2 carbon atoms and at least onetriple bond. Alkynyl can include any number of carbons, such as C₂,C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆,C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆. Examples of alkynyl groups include,but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl,isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl,isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl,3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl,2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynyl groups can be optionallysubstituted with one or more moieties selected from halo, hydroxy,amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, andcyano.

As used herein, the term “aryl” refers to an aromatic carbon ring systemhaving any suitable number of ring atoms and any suitable number ofrings. Aryl groups can include any suitable number of carbon ring atoms,such as, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, as well asfrom 6 to 10, 6 to 12, or 6 to 14 ring members. Aryl groups can bemonocyclic, fused to form bicyclic or tricyclic groups, or linked by abond to form a biaryl group. Representative aryl groups include phenyl,naphthyl and biphenyl. Other aryl groups include benzyl, having amethylene linking group. Some aryl groups have from 6 to 12 ringmembers, such as phenyl, naphthyl or biphenyl. Other aryl groups havefrom 6 to 10 ring members, such as phenyl or naphthyl. Some other arylgroups have 6 ring members, such as phenyl. Aryl groups can beoptionally substituted with one or more moieties selected from alkyl,alkenyl, alkynyl, haloalkyl, halo, hydroxy, amino, alkylamino, alkoxy,haloalkyl, carboxy, alkyl carboxylate, amido, nitro, oxo, and cyano.

As used herein, the term “cycloalkyl” refers to a saturated or partiallyunsaturated, monocyclic, fused bicyclic or bridged polycyclic ringassembly containing from 3 to 12 ring atoms, or the number of atomsindicated. Cycloalkyl can include any number of carbons, such as C₃₋₆,C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, and C₆₋₈. Saturated monocyclic cycloalkylrings include, for example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkylrings include, for example, norbornane, [2.2.2] bicyclooctane,decahydronaphthalene and adamantane. Cycloalkyl groups can also bepartially unsaturated, having one or more double or triple bonds in thering. Representative cycloalkyl groups that are partially unsaturatedinclude, but are not limited to, cyclobutene, cyclopentene, cyclohexene,cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene,cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene,and norbornadiene. Cycloalkyl groups can be optionally substituted withone or more moieties selected from alkyl, alkenyl, alkynyl, haloalkyl,halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, alkylcarboxylate, amido, nitro, oxo, and cyano.

As used herein, the term “heterocyclyl” refers to a saturated ringsystem having from 3 to 12 ring members and from 1 to 4 heteroatomsselected from N, O and S. Additional heteroatoms including, but notlimited to, B, Al, Si and P can also be present in a heterocycloalkylgroup. The heteroatoms can be oxidized to form moieties such as, but notlimited to, —S(O)— and —S(O)₂—. Heterocyclyl groups can include anynumber of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 4 to 6, or 4 to 7ring members. Any suitable number of heteroatoms can be included in theheterocyclyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2to 3, 2 to 4, or 3 to 4. Examples of heterocyclyl groups include, butare not limited to, aziridine, azetidine, pyrrolidine, piperidine,azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine(1,2-, 1,3- and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane(tetrahydropyran), oxepane, thiirane, thietane, thiolane(tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine,isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane,morpholine, thiomorpholine, dioxane, or dithiane. Heterocyclyl groupscan be optionally substituted with one or more moieties selected fromalkyl, alkenyl, alkynyl, haloalkyl, halo, hydroxy, amino, alkylamino,alkoxy, haloalkyl, carboxy, alkyl carboxylate, amido, nitro, oxo, andcyano.

As used herein, the term “heteroaryl” refers to a monocyclic or fusedbicyclic or tricyclic aromatic ring assembly containing 5 to 16 ringatoms, where from 1 to 5 of the ring atoms are a heteroatom such as N, Oor S. Additional heteroatoms including, but not limited to, B, Al, Siand P can also be present in a heteroaryl group. The heteroatoms can beoxidized to form moieties such as, but not limited to, —S(O)— and—S(O)₂—. Heteroaryl groups can include any number of ring atoms, suchas, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatomscan be included in the heteroaryl groups, such as 1, 2, 3, 4, or 5, or 1to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5.Heteroaryl groups can have from 5 to 8 ring members and from 1 to 4heteroatoms, or from 5 to 8 ring members and from 1 to 3 heteroatoms, orfrom 5 to 6 ring members and from 1 to 4 heteroatoms, or from 5 to 6ring members and from 1 to 3 heteroatoms. Examples of heteroaryl groupsinclude, but are not limited to, pyrrole, pyridine, imidazole, pyrazole,triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-,1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole,oxazole, and isoxazole. Heteroaryl groups can be optionally substitutedwith one or more moieties selected from alkyl, alkenyl, alkynyl,haloakyl, halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy,alkyl carboxylate, amido, nitro, oxo, and cyano.

As used herein, the term “alkoxy” refers to an alkyl group having anoxygen atom that connects the alkyl group to the point of attachment:i.e., alkyl-O—. As for alkyl group, alkoxy groups can have any suitablenumber of carbon atoms, such as C₁₋₆ or C₁₋₄. Alkoxy groups include, forexample, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy,iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. Alkoxy groupscan be optionally substituted with one or more moieties selected fromhalo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, and cyano.

As used herein, the term “alkylthio” refers to an alkyl group having asulfur atom that connects the alkyl group to the point of attachment:i.e., alkyl-S—. As for alkyl groups, alkylthio groups can have anysuitable number of carbon atoms, such as C₁₋₆ or C₁₋₄. Alkylthio groupsinclude, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy,2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc.Alkylthio groups can be optionally substituted with one or more moietiesselected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl,carboxy, amido, nitro, oxo, and cyano.

As used herein, the terms “halo” and “halogen” refer to fluorine,chlorine, bromine and iodine.

As used herein, the term “haloalkyl” refers to an alkyl moiety asdefined above substituted with at least one halogen atom.

As used herein, the term “alkylsilyl” refers to a moiety —SiR₃, whereinat least one R group is alkyl and the other R groups are H or alkyl. Thealkyl groups can be substituted with one more halogen atoms.

As used herein, the term “acyl” refers to a moiety —C(O)R, wherein R isan alkyl group.

As used herein, the term “oxo” refers to an oxygen atom that isdouble-bonded to a compound (i.e., O═).

As used herein, the term “carboxy” refers to a moiety —C(O)OH. Thecarboxy moiety can be ionized to form the carboxylate anion. “Alkylcarboxylate” refers to a moiety —C(O)OR, wherein R is an alkyl group asdefined herein.

As used herein, the term “amino” refers to a moiety —NR₃, wherein each Rgroup is H or alkyl.

As used herein, the term “amido” refers to a moiety —NRC(O)R or—C(O)NR₂, wherein each R group is H or alkyl.

As used herein, the term “organosilicon compound” means anorganometallic compound that contains a carbon-silicon (C—Si) bond.Typically, silicon atoms in organosilicon compounds are tetravalent andpossess a tetrahedral geometry. Compared to carbon-carbon bonds, C—Sibonds are typically longer and weaker, with slight polarization due torelatively higher carbon electronegativity. Organosilicon compoundscommonly have properties similar to other organic compounds, includingbeing colorless, hydrophobic, and flammable. Organosilicon compoundsfind use in a large number of industrial and commercial applications,including silicone compounds and silicon-derived products such ascoatings, adhesives, and sealants. Organosilicons can function asinsecticides and find utility in biotechnology applications.Non-limiting examples of biotechnology applications include antiviral,antiparasitic, and other drugs, as well as biocompatible coatings andcomponents for medical devices. Organosilicon compounds also findutility as reagents in various synthetic organic chemistry applications.

The terms “protein,” “peptide,” and “polypeptide” are usedinterchangeably herein to refer to a polymer of amino acid residues, oran assembly of multiple polymers of amino acid residues. The terms applyto amino acid polymers in which one or more amino acid residues are anartificial chemical mimic of a corresponding naturally occurring aminoacid, as well as to naturally occurring amino acid polymers andnon-naturally occurring amino acid polymers.

The term “amino acid” includes naturally-occurring α-amino acids andtheir stereoisomers, as well as unnatural (non-naturally occurring)amino acids and their stereoisomers. “Stereoisomers” of amino acidsrefers to mirror image isomers of the amino acids, such as L-amino acidsor D-amino acids. For example, a stereoisomer of a naturally-occurringamino acid refers to the mirror image isomer of the naturally-occurringamino acid, i.e., the D-amino acid.

Naturally-occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, y-carboxyglutamate and O-phosphoserine.Naturally-occurring α-amino acids include, without limitation, alanine(Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu),phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile),arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met),asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser),threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), andcombinations thereof. Stereoisomers of naturally-occurring a-amino acidsinclude, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys),D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine(D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg),D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine(D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser),D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine(D-Tyr), and combinations thereof.

Unnatural (non-naturally occurring) amino acids include, withoutlimitation, amino acid analogs, amino acid mimetics, synthetic aminoacids, N-substituted glycines, and N-methyl amino acids in either the L-or D-configuration that function in a manner similar to thenaturally-occurring amino acids. For example, “amino acid analogs” areunnatural amino acids that have the same basic chemical structure asnaturally-occurring amino acids, i.e., an a carbon that is bound to ahydrogen, a carboxyl group, an amino group, but have modified R (i.e.,side-chain) groups or modified peptide backbones, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. “Aminoacid mimetics” refer to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. For example, an L-aminoacid may be represented herein by its commonly known three letter symbol(e.g., Arg for L-arginine) or by an upper-case one-letter amino acidsymbol (e.g., R for L-arginine). A D-amino acid may be representedherein by its commonly known three letter symbol (e.g., D-Arg forD-arginine) or by a lower-case one-letter amino acid symbol (e.g., r forD-arginine).

With respect to amino acid sequences, one of skill in the art willrecognize that individual substitutions, additions, or deletions to apeptide, polypeptide, or protein sequence which alters, adds, or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. The chemically similar amino acid includes, withoutlimitation, a naturally-occurring amino acid such as an L-amino acid, astereoisomer of a naturally occurring amino acid such as a D-amino acid,and an unnatural amino acid such as an amino acid analog, amino acidmimetic, synthetic amino acid, N-substituted glycine, and N-methyl aminoacid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, substitutions may be madewherein an aliphatic amino acid (e.g., G, A, I, L, or V) is substitutedwith another member of the group. Similarly, an aliphaticpolar-uncharged group such as C, S, T, M, N, or Q, may be substitutedwith another member of the group; and basic residues, e.g., K, R, or H,may be substituted for one another. In some embodiments, an amino acidwith an acidic side chain, e.g., E or D, may be substituted with itsuncharged counterpart, e.g., Q or N, respectively; or vice versa. Eachof the following eight groups contains other exemplary amino acids thatare conservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)-   (see, e.g., Creighton, Proteins, 1993).

The term “oligonucleotide,” “nucleic acid,” “nucleotide,” or“polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleicacids (RNA) and polymers thereof in either single-, double- ormulti-stranded form. The term includes, but is not limited to, single-,double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNAhybrids, or a polymer comprising purine and/or pyrimidine bases or othernatural, chemically modified, biochemically modified, non-natural,synthetic or derivatized nucleotide bases. Unless specifically limited,the term encompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), orthologs, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985), and Rossolini et al., Mol.Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, and mRNA encoded by a gene.

The term “site-directed mutagenesis” refers to various methods in whichspecific changes are intentionally made introduced into a nucleotidesequence (i.e., specific nucleotide changes are introduced atpre-determined locations). Known methods of performing site-directedmutagenesis include, but are not limited to, PCR site-directedmutagenesis, cassette mutagenesis, whole plasmid mutagenesis, andKunkel's method.

The term “site-saturation mutagenesis,” also known as “saturationmutagenesis,” refers to a method of introducing random mutations atpredetermined locations with a nucleotide sequence, and is a methodcommonly used in the context of directed evolution (e.g., theoptimization of proteins (e.g., in order to enhance activity, stability,and/or stability), metabolic pathways, and genomes). In site-saturationmutagenesis, artificial gene sequences are synthesized using one or moreprimers that contain degenerate codons; these degenerate codonsintroduce variability into the position(s) being optimized. Each of thethree positions within a degenerate codon encodes a base such as adenine(A), cytosine (C), thymine (T), or guanine (G), or encodes a degenerateposition such as K (which can be G or T), M (which can be A or C), R(which can be A or G), S (which can be C or G), W (which can be A or T),Y (which can be C or T), B (which can be C, G, or T), D (which can be A,G, or T), H (which can be A, C, or T), V (which can be A, C, or G), or N(which can be A, C, G, or T). Thus, as a non-limiting example, thedegenerate codon NDT encodes an A, C, G, or T at the first position, anA, G, or T at the second position, and a T at the third position. Thisparticular combination of 12 codons represents 12 amino acids (Phe, Leu,Ile, Val, Tyr, His, Asn, Asp, Cys, Arg, Ser, and Gly). As anothernon-limiting example, the degenerate codon VHG encodes an A, C, or G atthe first position, an A, C, or T at the second position, and G at thethird position. This particular combination of 9 codons represents 8amino acids (Lys, Thr, Met, Glu, Pro, Leu, Ala, and Val). As anothernon-limiting example, the “fully randomized” degenerate codon NNNincludes all 64 codons and represents all 20 naturally-occurring aminoacids.

In some instances, a mixture of degenerate primers is used. A mixture ofdegenerate primers can contain any number of different degenerateprimers in any ratio. As a non-limiting example, a mixture of primerscontaining the NDT, VHG, and TGG primers can be used. Such a mixture cancontain, for example, an amount of each primer in a 12:9:1 ratio (e.g.,a NDT:VHG:TGG ratio of 12:9:1). Based on various considerations,non-limiting examples being desired redundancy, the desired presence ofstop codons, and/or desired amino acid characteristics (e.g., thepresence of nonpolar residues, charged residues, or small side chainresidues), different combinations of degenerate primers can be used.Considerations and methods for choosing optimal combinations ofdegenerate primers will be known to one of skill in the art.

The term “nucleotide sequence encoding a peptide” means the segment ofDNA involved in producing a peptide chain. The term can include regionspreceding and following the coding region (leader and trailer) involvedin the transcription/translation of a gene product and the regulation ofthe transcription/translation, as well as intervening sequences(introns) between individual coding segments (exons).

The term “homolog,” as used herein with respect to an original enzyme orgene of a first family or species, refers to distinct enzymes or genesof a second family or species which are determined by functional,structural or genomic analyses to be an enzyme or gene of the secondfamily or species which corresponds to the original enzyme or gene ofthe first family or species. Homologs most often have functional,structural, or genomic similarities. Techniques are known by whichhomologs of an enzyme or gene can readily be cloned using genetic probesand PCR. Identity of cloned sequences as homolog can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if theamino acid sequence encoded by a gene has a similar amino acid sequenceto that of the second gene. Alternatively, a protein has homology to asecond protein if the two proteins have “similar” amino acid sequences.Thus, the term “homologous proteins” is intended to mean that the twoproteins have similar amino acid sequences. In particular embodiments,the homology between two proteins is indicative of its shared ancestry,related by evolution.

III. Description of the Embodiments

A. Heme Proteins

In certain aspects, the present invention provides compositionscomprising one or more heme proteins that catalyze the formation oforganosilicon compounds from silicon-containing reagents and carbeneprecursors. In particular embodiments, the present invention providesheme protein variants comprising one or more amino acid mutationstherein that catalyze carbene insertion into silicon-hydrogen bonds,making organosilicon products with high stereoselectivity. In preferredembodiments, the heme protein variants of the present invention have theability to form carbon-silicon bonds efficiently, display increasedtotal turnover numbers, and/or demonstrate highly regio- and/orenantioselective product formation compared to the correspondingwild-type enzymes.

The terms “heme protein” and “heme enzyme” are used herein to includeany member of a group of proteins containing heme as a prosthetic group.Non-limiting examples of heme proteins include globins, cytochromes,oxidoreductases, any other protein containing a heme as a prostheticgroup, and combinations thereof. Heme-containing globins include, butare not limited to, hemoglobin, myoglobin, and combinations thereof.Heme-containing cytochromes include, but are not limited to, cytochromeP450, cytochrome b, cytochrome c1, cytochrome c, and combinationsthereof. Heme-containing oxidoreductases include, but are not limitedto, catalases, oxidases, oxygenases, haloperoxidases, peroxidases, andcombinations thereof. In some instances, the globin protein is fromMethylacidiphilum infernorum. In some other instances, the cytochromeP450 protein is a cytochrome P450 BM3 (CYP102A1) protein.

In certain instances, the heme proteins are metal-substituted hemeproteins containing protoporphyrin IX or other porphyrin moleculescontaining non-native cofactors (i.e., metals other than iron),including, but not limited to, cobalt, rhodium, copper, ruthenium,iridium, and manganese, which are active carbon-silicon bond formationcatalysts.

In some embodiments, the heme protein is a member of one of the enzymeclasses set forth in Table 1. In other embodiments, the heme protein isa variant or homolog of a member of one of the enzyme classes set forthin Table 1. In yet other embodiments, the heme protein comprises orconsists of the heme domain of a member of one of the enzyme classes setforth in Table 1 or a fragment thereof (e.g., a truncated heme domain)that is capable of carrying out the carbene insertion reactionsdescribed herein.

TABLE 1 Heme enzymes identified by their enzyme classification number(EC number) and classification name EC Number Name 1.1.2.3 L-lactatedehydrogenase 1.1.2.6 polyvinyl alcohol dehydrogenase (cytochrome)1.1.2.7 methanol dehydrogenase (cytochrome c) 1.1.5.5 alcoholdehydrogenase (quinone) 1.1.5.6 formate dehydrogenase-N: 1.1.9.1 alcoholdehydrogenase (azurin): 1.1.99.3 gluconate 2-dehydrogenase (acceptor)1.1.99.11 fructose 5-dehydrogenase 1.1.99.18 cellobiose dehydrogenase(acceptor) 1.1.99.20 alkan-1-ol dehydrogenase (acceptor) 1.2.1.70glutamyl-tRNA reductase 1.2.3.7 indole-3-acetaldehyde oxidase 1.2.99.3aldehyde dehydrogenase (pyrroloquinoline-quinone) 1.3.1.6 fumaratereductase (NADH): 1.3.5.1 succinate dehydrogenase (ubiquinone) 1.3.5.4fumarate reductase (menaquinone) 1.3.99.1 succinate dehydrogenase1.4.9.1 methylamine dehydrogenase (amicyanin) 1.4.9.2. aralkylaminedehydrogenase (azurin) 1.5.1.20 methylenetetrahydrofolate reductase[NAD(P)H] 1.5.99.6 spermidine dehydrogenase 1.6.3.1 NAD(P)H oxidase1.7.1.1 nitrate reductase (NADH) 1.7.1.2 Nitrate reductase [NAD(P)H]1.7.1.3 nitrate reductase (NADPH) 1.7.1.4 nitrite reductase [NAD(P)H]1.7.1.14 nitric oxide reductase [NAD(P), nitrous oxide-forming] 1.7.2.1nitrite reductase (NO-forming) 1.7.2.2 nitrite reductase (cytochrome;ammonia-forming) 1.7.2.3 trimethylamine-N-oxide reductase (cytochrome c)1.7.2.5 nitric oxide reductase (cytochrome c) 1.7.2.6 hydroxylaminedehydrogenase 1.7.3.6 hydroxylamine oxidase (cytochrome) 1.7.5.1 nitratereductase (quinone) 1.7.5.2 nitric oxide reductase (menaquinol) 1.7.6.1nitrite dismutase 1.7.7.1 ferredoxin-nitrite reductase 1.7.7.2ferredoxin-nitrate reductase 1.7.99.4 nitrate reductase 1.7.99.8hydrazine oxidoreductase 1.8.1.2 sulfite reductase (NADPH) 1.8.2.1sulfite dehydrogenase 1.8.2.2 thiosulfate dehydrogenase 1.8.2.3sulfide-cytochrome-c reductase (flavocytochrome c) 1.8.2.4 dimethylsulfide:cytochrome c2 reductase 1.8.3.1 sulfite oxidase 1.8.7.1 sulfitereductase (ferredoxin) 1.8.98.1 CoB-CoM heterodisulfide reductase1.8.99.1 sulfite reductase 1.8.99.2 adenylyl-sulfate reductase 1.8.99.3hydrogensulfite reductase 1.9.3.1 cytochrome-c oxidase 1.9.6.1 nitratereductase (cytochrome) 1.10.2.2 ubiquinol-cytochrome-c reductase1.10.3.1 catechol oxidase 1.10.3.B1 caldariellaquinol oxidase(H+-transporting) 1.10.3.3 L-ascorbate oxidase 1.10.3.9 photosystem II1.10.3.10 ubiquinol oxidase (H+-transporting) 1.10.3.11 ubiquinoloxidase 1.10.3.12 menaquinol oxidase (H+-transporting) 1.10.9.1plastoquinol-plastocyanin reductase 1.11.1.5 cytochrome-c peroxidase1.11.1.6 catalase 1.11.1.7 peroxidase 1.11.1.B2 chloride peroxidase(vanadium-containing) 1.11.1.B7 bromide peroxidase (heme-containing)1.11.1.8 iodide peroxidase 1.11.1.10 chloride peroxidase 1.11.1.11L-ascorbate peroxidase 1.11.1.13 manganese peroxidase 1.11.1.14 ligninperoxidase 1.11.1.16 versatile peroxidase 1.11.1.19 dye decolorizingperoxidase 1.11.1.21 catalase-peroxidase 1.11.2.1 unspecificperoxygenase 1.11.2.2 myeloperoxidase 1.11.2.3 plant seed peroxygenase1.11.2.4 fatty-acid peroxygenase 1.12.2.1 cytochrome-c3 hydrogenase1.12.5.1 hydrogen:quinone oxidoreductase 1.12.99.6 hydrogenase(acceptor) 1.13.11.9 2,5-dihydroxypyridine 5,6-dioxygenase 1.13.11.11tryptophan 2,3-dioxygenase 1.13.11.49 chlorite O2-lyase 1.13.11.50acetylacetone-cleaving enzyme 1.13.11.52 indoleamine 2,3-dioxygenase1.13.11.60 linoleate 8R-lipoxygenase 1.13.99.3 tryptophan 2′-dioxygenase1.14.11.9 flavanone 3-dioxygenase 1.14.12.17 nitric oxide dioxygenase1.14.13.39 nitric-oxide synthase (NADPH dependent) 1.14.13.17cholesterol 7alpha-monooxygenase 1.14.13.41 tyrosine N-monooxygenase1.14.13.70 sterol 14alpha-demethylase 1.14.13.71 N-methylcoclaurine3′-monooxygenase 1.14.13.81 magnesium-protoporphyrin IX monomethyl ester(oxidative) cyclase 1.14.13.86 2-hydroxyisoflavanone synthase 1.14.13.98cholesterol 24-hydroxylase 1.14.13.119 5-epiaristolochene1,3-dihydroxylase 1.14.13.126 vitamin D3 24-hydroxylase 1.14.13.129beta-carotene 3-hydroxylase 1.14.13.141 cholest-4-en-3-one26-monooxygenase 1.14.13.142 3-ketosteroid 9alpha-monooxygenase1.14.13.151 linalool 8-monooxygenase 1.14.13.156 1,8-cineole2-endo-monooxygenase 1.14.13.159 vitamin D 25-hydroxylase 1.14.14.1unspecific monooxygenase 1.14.15.1 camphor 5-monooxygenase 1.14.15.6cholesterol monooxygenase (side-chain-cleaving) 1.14.15.8 steroid15beta-monooxygenase 1.14.15.9 spheroidene monooxygenase 1.14.18.1tyrosinase 1.14.19.1 stearoyl-CoA 9-desaturase 1.14.19.3 linoleoyl-CoAdesaturase 1.14.21.7 biflaviolin synthase 1.14.99.1prostaglandin-endoperoxide synthase 1.14.99.3 heme oxygenase 1.14.99.9steroid 17alpha-monooxygenase 1.14.99.10 steroid 21-monooxygenase1.14.99.15 4-methoxybenzoate monooxygenase (O-demethylating) 1.14.99.45carotene epsilon-monooxygenase 1.16.5.1 ascorbate ferrireductase(transmembrane) 1.16.9.1 iron:rusticyanin reductase 1.17.1.4 xanthinedehydrogenase 1.17.2.2 lupanine 17-hydroxylase (cytochrome c) 1.17.99.14-methylphenol dehydrogenase (hydroxylating) 1.17.99.2 ethylbenzenehydroxylase 1.97.1.1 chlorate reductase 1.97.1.9 selenate reductase2.7.7.65 diguanylate cyclase 2.7.13.3 histidine kinase 3.1.4.52cyclic-guanylate-specific phosphodiesterase 4.2.1.B9 colneleicacid/etheroleic acid synthase 4.2.1.22 Cystathionine beta-synthase4.2.1.92 hydroperoxide dehydratase 4.2.1.212 colneleate synthase4.3.1.26 chromopyrrolate synthase 4.6.1.2 guanylate cyclase 4.99.1.3sirohydrochlorin cobaltochelatase 4.99.1.5 aliphatic aldoximedehydratase 4.99.1.7 phenylacetaldoxime dehydratase 5.3.99.3prostaglandin-E synthase 5.3.99.4 prostaglandin-I synthase 5.3.99.5Thromboxane-A synthase 5.4.4.5 9,12-octadecadienoate 8-hydroperoxide8R-isomerase 5.4.4.6 9,12-octadecadienoate 8-hydroperoxide 8S-isomerase6.6.1.2 cobaltochelatase

In particular embodiments, the heme protein is a variant or a fragmentthereof (e.g., a truncated variant containing the heme domain)comprising at least one mutation, e.g., a mutation at the axial positionof the heme coordination site. In some instances, the mutation is asubstitution of the native residue with Ala, Asp, Arg, Asn, Cys, Glu,Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Valat the axial position. In certain instances, the mutation is asubstitution of Met with any other amino acid such as Asp or Glu at theaxial position.

In certain embodiments, the heme protein, variant thereof, or homologthereof comprises or consists of the same number of amino acid residuesas the wild-type protein (e.g., a full-length polypeptide). In someinstances, the heme protein, variant thereof, or homolog thereofcomprises or consists of a fragment of the full-length protein (e.g.,Rma cyt c amino acid sequence set forth in SEQ ID NO:1).

In some embodiments, the heme enzyme comprises a globin enzyme. Globinsare a superfamily of globular heme proteins that are typically involvedin the transport and binding of oxygen. A characteristic of globins is athree-dimensional fold consisting of eight alpha helices, although someglobins have additional terminal helix extensions. Globins can bedivided into three groups: single-domain globins, flavohemoglobins (notobserved in archaea), and globin-coupled sensors (not observed ineukaryotes). All three groups are observed in bacteria. Globin proteinsinclude hemoglobin, myoglobin, neuroglobin, cytoglobin, erythrocruorin,leghemoglobin, non-symbiotic hemoglobin, flavohemoglobins (one group ofchimeric globins), globin E, globin-coupled sensors (another group ofchimeric globins), protoglobin, truncated 2/2 globin, HbN, cyanoglobin,HbO, and Glb3.

In other embodiments, the heme enzyme comprises an oxidoreductase.Oxidoreductases are enzymes that catalyze the transfer of electrons froma reductant (i.e., an electron donor) to an oxidant (i.e., an electronacceptor) and are divided into 22 subclasses. Oxidoreductases typicallyutilize NADP or NAD+ as a cofactor. EC 1.1 oxidoreductases (alcoholoxidoreductases) act on the CH—OH group of donors. EC 1.2oxidoreductases act on the aldehyde or oxo group of donors. EC 1.3oxidoreductases (CH—CH oxidoreductases) act on the CH—CH group ofdonors. EC 1.4 oxidoreductases (amino acid oxidoreductases, monoamineoxidase) act on the CH—NH₂ group of donors. EC 1.5 oxidoreductases acton the CH—NH group of donors. EC 1.6 oxidoreductases act on NADH orNADPH. EC 1.7 oxidoreductases act on other nitrogenous compounds asdonors. EC 1.8 oxidoreductases act on a sulfur group of donors. EC 1.9oxidoreductases act on a heme group of donors. EC 1.10 oxidoreductasesact on diphenols and related substances as donors. EC 1.11oxidoreductases (peroxidases) act on peroxide as an acceptor. EC 1.12oxidoreductases act on hydrogen as donors. EC 1.13 oxidoreductases(oxygenases) act on single donors with incorporation of molecularoxygen. EC 1.14 oxidoreductases act on paired donors with incorporationof molecular oxygen. EC 1.15 oxidoreductases act on superoxide radicalsas acceptors. EC 1.16 oxidoreductases oxidize metal ions. EC 1.17oxidoreductases act on CH or CH2 groups. EC 1.18 oxidoreductases act oniron-sulfur proteins as donors. EC 1.19 oxidoreductases act on reducedflavodoxin as a donor. EC 1.20 oxidoreductases act on phosphorous orarsenic in donors. EC 1.21 oxidoreductases act on X—H and Y—H to form anX—Y bond. Enzyme classification number 1.97 includes otheroxidoreductases that do not fit into any of the aforementionedsubclasses. Haloperoxidases are peroxidases that mediate halideoxidation by hydrogen peroxide (EC 1.11.1). Catalases catalyze thedecomposition of hydrogen peroxide to oxygen and water (EC 1.11.1.6).

In some embodiments, the heme enzyme comprises a cytochrome. Cytochromesare a class of heme proteins that are found in bacteria, as well asmitochondria and chloroplasts of eukaryotic organisms, and are typicallyassociated with membranes. Cytochromes typically function in oxidativephosphorylation as components of electron transport chain systems.Cytochromes can be classified by spectroscopy, or by features such asthe structure of the heme group, inhibitor sensitivity, or reductionpotential. Three of the cytochromes, cytochromes a, b, and d, areclassified by their prosthetic group (the prosthetic groups consistingof heme a, heme b, and tetrapyrrolic chelate of iron, respectively).Unlike the aforementioned cytochromes, cytochrome c is not defined interms of its heme group. Cytochrome f, which performs similar functionsto cytochrome c₁ but has a different structure, is sometimes regarded asa type of cytochrome c. Cytochrome P450 proteins form a distinct familyof cytochromes.

In bacteria, mitochondria, and chloroplasts, various cytochromes formdifferent combinations to that perform different functions. Cytochromesa and a₃ combine to form cytochrome c oxidase (also known as ComplexIV), which is the last enzyme in the respiratory chain of bacteria andmitochondria. Cytochromes b and c₁ combine to form coenzyme Q—cytochromec reductase—the third complex in the electron transport chain.Cytochromes b₆ and f combine to form plastoquinol-plastocyaninreductase, which is found in the chloroplasts of plants, cyanobacteriaand green algae and functions in photosynthesis.

Cytochrome P450 enzymes constitute a large superfamily of heme-thiolateproteins involved in the metabolism of a wide variety of both exogenousand endogenous compounds. Usually, they act as the terminal oxidase inmulticomponent electron transfer chains, such as P450-containingmonooxygenase systems. Members of the cytochrome P450 enzyme familycatalyze myriad oxidative transformations, including, e.g.,hydroxylation, epoxidation, oxidative ring coupling, heteroatom release,and heteroatom oxygenation (E. M. Isin et al., Biochim. Biophys. Acta1770, 314 (2007)). The active site of these enzymes contains anFe^(III)-protoporphyrin IX cofactor (heme) ligated proximally by aconserved cysteine thiolate (M. T. Green, Current Opinion in ChemicalBiology 13, 84 (2009)). The remaining axial iron coordination site isoccupied by a water molecule in the resting enzyme, but during nativecatalysis, this site is capable of binding molecular oxygen. In thepresence of an electron source, typically provided by NADH or NADPH froman adjacent fused reductase domain or an accessory cytochrome P450reductase enzyme, the heme center of cytochrome P450 activates molecularoxygen, generating a high valent iron(IV)-oxo porphyrin cation radicalspecies intermediate and a molecule of water.

Cytochrome P450 BM3 (CYP102A1) proteins are found in the soil bacteriumBacillus megaterium and catalyze the NADPH-dependent hydroxylation oflong-chain fatty acids at the ω-1 through ω-3 positions. Unlike mostother cytochrome P450 proteins, cytochrome P450 BM3 proteins are anatural fusion between the cytochrome P450 domain and an electrondonating cofactor. Thus, cytochrome P450 BM3 proteins are useful in anumber of biotechnological applications.

Cytochrome c proteins are a superfamily of proteins that have one ormore covalently bound heme prosthetic groups (i.e., heme c groups).Generally, the heme groups are bound to the protein by one, or moretypically two, thioether bonds involving sulphydryl groups of cysteineresidues. This superfamily of proteins possesses a characteristic CXXCHamino acid motif that binds heme, wherein X can be any amino acid. Thefifth heme iron ligand is provided by a histidine residue. Cytochrome cproteins possess a wide range of characteristics, enabling them tofunction in a large number of redox processes.

Cytochrome c is highly conserved across the spectrum of species.Non-limiting examples of cytochrome c amino acid sequences (encoded bythe CYCS gene) can be found in NCBI Reference Sequence No.NM_018947.5→NP_061820.1 (human), NCBI Reference Sequence No.NM_007808.4→NP_031834.1 (mouse), and NCBI Reference No. ACA83734.1(Rhodothermus marinus unprocessed; SEQ ID NO:2).

Cytochrome c proteins fall into one of four classes. Class I containssoluble, low spin single domain C-type cytochromes. There are at leastsix subclasses of Class 1 cytochrome c proteins that are found inprokaryotes including Desulfovibrio desulfuricans, Rhodospirillumrubrum, Rhodopila globiformis, and Rhodothermus marinus (Rma). Class Iproteins have a single heme that is attached near the N-terminus of thepolypeptide, with a methionine residue being the sixth iron coordinationsite. Class II contains higher spin-state cytochromes c, with the hemeprosthetic group being attached closer to the C-terminus. Class IIIcontains cytochromes with multiple heme groups. These proteins havelower redox potentials compared to the other three classes. Class IVcontains more complex proteins having higher molecular weights. Class IVproteins contain heme c as well as other prosthetic groups.

In some embodiments, the cytochrome c protein is selected from the groupconsisting of Rhodothermus marinus (Rma) cytochrome c, Rhodopilaglobiformis cytochrome c, Hydrogenobacter thermophilus cytochrome c,Saccharomyces cerevisiae cytochrome c, horse heart cytochrome c, bovineheart cytochrome c, and a combination thereof.

In preferred embodiments, the cytochrome c protein variant comprises amutation at one or more of the conserved residues of the correspondingwild-type sequence that serve as heme axial ligands. In certainpreferred embodiments, the cytochrome c protein variant comprises one ormore mutations at one or more conserved residues of the correspondingwild-type sequences that reside near (e.g., about 7 Å) the heme center.As a non-limiting example, an axial variant of Rma cyt c can comprise aM100 mutation relative to the amino acid sequence set forth in SEQ IDNO:1. In some instances, the mutation is an M100D or M100E mutation. Asanother other non-limiting example, a variant of Rma cyt c can comprisea mutation at V75 relative to the amino acid sequence set forth in SEQID NO:1. In some instances, the mutation is a V75T mutation. As anotherother non-limiting example, a variant of Rma cyt c can comprise amutation at M103 relative to the amino acid sequence set forth in SEQ IDNO:1. In some instances, the mutation is an M103E mutation.

In some other embodiments, the Rma cyt c protein variant comprises acombination of mutations (e.g., the Rma cyt c protein variant is a V75M100 mutant, a V75 M103 mutant, an M100 M103 mutant, or a V75 M100 M103mutant, relative to the amino acid sequence set forth in SEQ ID NO:1).In some instances, the Rma cyt c protein variant is a V75T M100D or V75TM100E mutant relative to the amino acid sequence set forth in SEQ IDNO:1. In other instances, the Rma cyt c protein variant is a V75T M103Emutant relative to the amino acid sequence set forth in SEQ ID NO:1. Insome instances, the Rma cyt c protein variant is an M100D M103E or M100Eor M103E mutant relative to the amino acid sequence set forth in SEQ IDNO:1. In other instances, the Rma cyt c protein variant is a V75T M100DM103E or V75T M100E M103E mutant relative to the amino acid sequence setforth in SEQ ID NO:1.

In some embodiments, the heme protein comprises an amino acid sequencethat has about 70% or greater (e.g., about 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity tothe amino acid sequence set forth in SEQ ID NO:1. In other embodiments,the heme protein comprises an amino acid sequence that has about 80% orgreater (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity tothe amino acid sequence set forth in SEQ ID NO:1. In particularembodiments, the heme protein comprises an amino acid sequence that hasabout 90% or greater (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identity to the amino acid sequence set forth inSEQ ID NO:1. In some instances, the heme protein comprises an amino acidsequence that is about 95%, 96,%, 97%, 98%, 99%, or 100% identical tothe amino acid sequence set forth in SEQ ID NO:1.

In other embodiments, the heme protein comprises an amino acid sequencethat contains between about 5 and 124 (e.g., about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124) of theamino acids in SEQ ID NO:1. The amino acids may be contiguous, orseparated by any number of amino acids.

In some embodiments, the heme protein comprises an amino acid sequencethat has about 70% or greater (e.g., about 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity tothe amino acid sequence set forth in SEQ ID NO:2. In other embodiments,the heme protein comprises an amino acid sequence that has about 80% orgreater (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity tothe amino acid sequence set forth in SEQ ID NO:2. In particularembodiments, the heme protein comprises an amino acid sequence that hasabout 90% or greater (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identity to the amino acid sequence set forth inSEQ ID NO:2. In some instances, the heme protein comprises an amino acidsequence that is about 95%, 96,%, 97%, 98%, 99%, or 100% identical tothe amino acid sequence set forth in SEQ ID NO:2.

In other embodiments, the heme protein comprises an amino acid sequencethat contains between about 5 and 152 (e.g., about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, or 152) of theamino acids in SEQ ID NO:2. The amino acids may be contiguous, orseparated by any number of amino acids.

In certain embodiments, the conserved residue in a heme protein ofinterest that serves as the heme axial ligand can be identified bylocating the segment of the DNA sequence in the corresponding cytochromec gene which encodes the conserved residue. In some instances, this DNAsegment is identified through detailed mutagenesis studies in aconserved region of the protein. In other instances, the conservedresidue is identified through crystallographic study.

In situations where detailed mutagenesis studies and crystallographicdata are not available for a heme protein of interest, the axial ligandmay be identified through phylogenetic study. Due to the similarities inamino acid sequence within families of heme proteins (e.g., cytochrome cproteins), standard protein alignment algorithms may show a phylogeneticsimilarity between a heme protein for which crystallographic ormutagenesis data exist and a new heme protein for which such data do notexist. Thus, the polypeptide sequences of the present invention forwhich the heme axial ligand is known can be used as a “query sequence”to perform a search against a specific new heme protein of interest or adatabase comprising heme protein sequences to identify the heme axialligand. Such analyses can be performed using the BLAST programs (see,e.g., Altschul et al., J Mol Biol. 215(3):403-10(1990)). Software forperforming BLAST analyses publicly available through the National Centerfor Biotechnology Information (ncbi.nlm.nih.gov). BLASTP is used foramino acid sequences.

Exemplary parameters for performing amino acid sequence alignments toidentify the heme axial ligand in a heme protein of interest using theBLASTP algorithm include E value=10, word size=3, Matrix=Blosum62, Gapopening=11, gap extension=1, and conditional compositional score matrixadjustment. Those skilled in the art will know what modifications can bemade to the above parameters, e.g., to either increase or decrease thestringency of the comparison and/or to determine the relatedness of twoor more sequences.

In certain embodiments, mutations can be introduced into the target geneusing standard cloning techniques (e.g., site-directed mutagenesis,site-saturated mutagenesis) or by gene synthesis to produce the hemeproteins, fragments thereof, variants thereof, or homologs thereof ofthe present invention.

In some embodiments, the heme protein, fragment thereof, variantthereof, or homolog thereof is recombinantly expressed and optionallyisolated and/or purified for carrying out the in vitro silicon-hydrogencarbene insertion reactions of the present invention. In otherembodiments, the heme protein, fragment thereof, variant thereof, orhomolog thereof is expressed in whole cells such as bacterial cells,archaeal cells, yeast cells, fungal cells, insect cells, plant cells, ormammalian cells, and these cells are used for carrying out the in vivosilicon-hydrogen carbene insertion reactions of the present invention.The wild-type or mutated gene can be expressed in a whole cell using anexpression vector under the control of an inducible promoter or by meansof chromosomal integration under the control of a constitutive promoter.Silicon-hydrogen carbene insertion activity can be screened in vivo orin vitro by following product formation by GC or HPLC.

Suitable bacterial host cells include, but are not limited to, BL21 E.coli, DE3 strain E. coli, E. coli M15, DH5α, DH10β, HB101, T7 ExpressCompetent E. coli (NEB), B. subtilis cells, Pseudomonas fluorescenscells, and cyanobacterial cells such as Chlamydomonas reinhardtii cellsand Synechococcus elongates cells. Non-limiting examples of archaealhost cells include Pyrococcus furiosus, Metallosphera sedula,Thermococcus litoralis, Methanobacterium thermoautotrophicum,Methanococcus jannaschii, Pyrococcus abyssi, Sulfolobus solfataricus,Pyrococcus woesei, Sulfolobus shibatae, and variants thereof. Fungalhost cells include, but are not limited to, yeast cells from the generaSaccharomyces (e.g., S. cerevisiae), Pichia (P. Pastoris), Kluyveromyces(e.g., K. lactis), Hansenula and Yarrowia, and filamentous fungal cellsfrom the genera Aspergillus, Trichoderma, and Myceliophthora. Suitableinsect host cells include, but are not limited to, Sf9 cells fromSpodoptera frugiperda, Sf21 cells from Spodoptera frugiperda, Hi-Fivecells, BTI-TN-5B1-4 Trichophusia ni cells, and Schneider 2 (S2) cellsand Schneider 3 (S3) cells from Drosophila melanogaster. Non-limitingexamples of mammalian host cells include HEK293 cells, HeLa cells, CHOcells, COS cells, Jurkat cells, NS0 hybridoma cells, baby hamster kidney(BHK) cells, MDCK cells, NIH-3T3 fibroblast cells, and any otherimmortalized cell line derived from a mammalian cell. Non-limitingexamples of plant host cells include those from tobacco, tomato, potato,maize, rice, lettuce, and spinach. In general, cells from plants thathave short generation times and/or yield reasonable biomass withstandard cultivation techniques are preferable.

In certain embodiments, the present invention provides the hemeproteins, fragments thereof, variants thereof, or homologs thereof, suchas the cytochrome c variants described herein that are activesilicon-hydrogen carbene insertion catalysts, inside living cells. As anon-limiting example, bacterial cells (e.g., E. coli) can be used ashost whole cell catalysts for the in vivo silicon-hydrogen carbeneinsertion reactions of the present invention, although any number ofhost whole cells may be used, including but not limited to the hostcells described herein. In some embodiments, host whole cell catalystscontaining heme proteins, fragments thereof, variants thereof, orhomologs thereof are found to significantly enhance the total turnovernumber (TTN) compared to the in vitro reactions using isolated hemeproteins, fragments thereof, variants thereof, or homologs thereof.

The expression vector comprising a nucleic acid sequence that encodes aheme protein, fragment thereof, variant thereof, or homolog thereof ofthe invention can be a viral vector, a plasmid, a phage, a phagemid, acosmid, a fosmid, a bacteriophage (e.g., a bacteriophage P1-derivedvector (PAC)), a baculovirus vector, a yeast plasmid, or an artificialchromosome (e.g., bacterial artificial chromosome (BAC), a yeastartificial chromosome (YAC), a mammalian artificial chromosome (MAC),and human artificial chromosome (HAC)). Expression vectors can includechromosomal, non-chromosomal, and synthetic DNA sequences. Equivalentexpression vectors to those described herein are known in the art andwill be apparent to the ordinarily skilled artisan.

The expression vector can include a nucleic acid sequence encoding aheme protein, fragment thereof, variant thereof, or homolog thereof thatis operably linked to a promoter, wherein the promoter comprises aviral, bacterial, archaeal, fungal, insect, plant, or mammalianpromoter. In certain embodiments, the promoter is a constitutivepromoter. In some embodiments, the promoter is an inducible promoter. Inother embodiments, the promoter is a tissue-specific promoter or anenvironmentally regulated or a developmentally regulated promoter.

In some embodiments, the nucleic acid sequence encodes a heme proteinthat comprises an amino acid sequence that has about 70% or greater(e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence setforth in SEQ ID NO:1. In other embodiments, the nucleic acid sequenceencodes a heme protein that comprises an amino acid sequence that hasabout 80% or greater (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identity to the amino acid sequence set forth in SEQ ID NO:1. Inparticular embodiments, the nucleic acid sequence encodes a heme proteinthat comprises an amino acid sequence that has about 90% or greater(e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identity to the amino acid sequence set forth in SEQ ID NO:1. In someinstances, the nucleic acid sequence encodes a heme protein thatcomprises an amino acid sequence that is about 95%, 96,%, 97%, 98%, 99%,or 100% identical to the amino acid sequence set forth in SEQ ID NO:1.

In other embodiments, the nucleic acid sequence encodes a heme proteinthat comprises an amino acid sequence that contains between about 5 and124 (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, or 124) of the amino acids in SEQ ID NO:1. Theamino acids may be contiguous, or separated by any number of aminoacids.

In some embodiments, the nucleic acid sequence encodes a heme proteinthat comprises an amino acid sequence that has about 70% or greater(e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence setforth in SEQ ID NO:2. In other embodiments, the nucleic acid sequenceencodes a heme protein that comprises an amino acid sequence that hasabout 80% or greater (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identity to the amino acid sequence set forth in SEQ ID NO:2. Inparticular embodiments, the nucleic acid sequence encodes a heme proteinthat comprises an amino acid sequence that has about 90% or greater(e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identity to the amino acid sequence set forth in SEQ ID NO:2. In someinstances, the nucleic acid sequence encodes a heme protein thatcomprises an amino acid sequence that is about 95%, 96,%, 97%, 98%, 99%,or 100% identical to the amino acid sequence set forth in SEQ ID NO:2.

In other embodiments, the nucleic acid sequence encodes a heme proteinthat comprises an amino acid sequence that contains between about 5 and152 (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, or 152) of the amino acids in SEQ ID NO:2. Theamino acids may be contiguous, or separated by any number of aminoacids.

It is understood that affinity tags may be added to the N- and/orC-terminus of a heme protein, fragment thereof, variant thereof, orhomolog thereof expressed using an expression vector to facilitateprotein purification. Non-limiting examples of affinity tags includemetal binding tags such as His6-tags and other tags such as glutathioneS-transferase (GST).

Non-limiting expression vectors for use in bacterial host cells includepCWori, pET vectors such as pET22 (EMD Millipore), pBR322 (ATCC37017),pQE™ vectors (Qiagen), pBluescript™ vectors (Stratagene), pNH vectors,lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T(Pharmacia), pRSET, pCR-TOPO vectors, pET vectors, pSyn_1 vectors,pChlamy_1 vectors (Life Technologies, Carlsbad, Calif.), pGEM1 (Promega,Madison, Wis.), and pMAL (New England Biolabs, Ipswich, Mass.).Non-limiting examples of expression vectors for use in eukaryotic hostcells include pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40(Pharmacia), pcDNA3.3, pcDNA4/TO, pcDNA6/TR, pLenti6/TR, pMT vectors(Life Technologies), pKLAC1 vectors, pKLAC2 vectors (New EnglandBiolabs), pQE™ vectors (Qiagen), BacPak baculoviral vectors, pAdeno-X™adenoviral vectors (Clontech), and pBABE retroviral vectors. Any othervector may be used as long as it is replicable and viable in the hostcell.

In some embodiments, the heme protein, homolog, variant, or fragmentthereof has a turnover frequency (TOF) between about 1 min⁻¹ and 10min⁻¹ (e.g., about 1 min⁻¹, 1.5 min⁻¹, 2 min⁻¹, 2.5 min⁻¹, 3 min⁻¹, 3.5min⁻¹, 4 min⁻¹, 4.5 min⁻¹, 5 min⁻¹, 5.5 min⁻¹, 6 min⁻¹, 6.5 min⁻¹, 7min⁻¹, 7.5 min⁻¹, 8 min⁻¹, 8.5 min⁻¹, 9 min⁻¹, 9.5 min⁻¹, or 10 min⁻¹).In other embodiments, the TOF is between about 10 min⁻¹ and 100 min⁻¹(e.g., about 10 min⁻¹, 11 min⁻¹, 12 min⁻¹, 13 min⁻¹, 14 min⁻¹, 15 min⁻¹,16 min⁻¹, 17 min⁻¹, 18 min⁻¹, 19 min⁻¹, 20 min⁻¹, 21 min⁻¹, 22 min⁻¹, 23min⁻¹, 24 min⁻¹, 25 min⁻¹, 26 min⁻¹, 27 min⁻¹, 28 min⁻¹, 29 min⁻¹, 30min⁻¹, 31 min⁻¹, 32 min⁻¹, 33 min⁻¹, 34 min⁻¹, 35 min⁻¹, 36 min⁻¹, 37min⁻¹, 38 min⁻¹, 39 min⁻¹, 40 min⁻¹, 41 min⁻¹, 42 min⁻¹, 43 min⁻¹, 44min⁻¹, 45 min⁻¹, 46 min⁻¹, 47 min⁻¹, 48 min⁻¹, 49 min⁻¹, 50 min⁻¹, 55min⁻¹, 60 min⁻¹, 65 min⁻¹, 70 min⁻¹, 75 min⁻¹, 80 min⁻¹, 85 min⁻¹, 90min⁻¹, 95 min⁻¹, or 100 min⁻¹). In other instances, the TOF is greaterthan about 100 min⁻¹ to 1,000 min⁻¹ (e.g., greater than about 100 min⁻¹,150 min⁻¹, 200 min⁻¹, 250 min⁻¹, 300 min⁻¹, 350 min⁻¹, 400 min⁻¹, 450min⁻¹, 500 min⁻¹, 550 min⁻¹, 600 min⁻¹, 650 min⁻¹, 700 min⁻¹, 750 min⁻¹,800 min⁻¹, 850 min⁻¹, 900 min⁻¹, 950 min⁻¹, 1,000 min⁻¹, or more). Insome instances, the TOF is greater than about 10 min⁻¹. In otherinstances, the TOF is greater than about 45 min⁻¹.

In other embodiments, the heme protein, homolog, variant, or fragmentthereof has a total turnover number (TTN), which refers to the maximumnumber of molecules of a substrate that the protein can convert beforebecoming inactivated, of between about 1 and 100 (e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100). In someother embodiments, the TTN is between about 100 and 1,000 (e.g., about100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, or 1,000). In some embodiments, the TTN is betweenabout 1,000 and 2,000 (e.g., about 1,000, 1,050, 1,100, 1,150, 1,200,1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,550, 1,600, 1,650, 1,700,1,750, 1,800, 1,850, 1,900, 1,950 or 2,000). In other embodiments, theTTN is at least about 2,000 (e.g., at least about 2,000, 2,500, 3,000,3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000,8,500, 9,000, 9,500, or 10,000). In some instances, the TTN is greaterthan about 70. In other instances, the TTN is greater than about 1,800.

In some embodiments, the heme protein variant or fragment thereof hasenhanced activity of at least about 1.5 to 2,000 fold (e.g., at leastabout 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050,1,100, 1,150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,550,1,600, 1,650, 1,700, 1,750, 1,800, 1,850, 1,900, 1,950, 2,000, or more)fold compared to the corresponding wild-type heme protein.

In some embodiments, activity is expressed in terms of turnoverfrequency (TOF). In particular embodiments, the TOF of the heme proteinvariant or fragment thereof is at least about 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 fold higher than thecorresponding wild-type protein.

In other instances, activity is expressed in terms of total turnovernumber (TTN). In particular instances, the TTN of the theme proteinvariant or fragment thereof is about least about 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,1,050, 1,100, 1,150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500,1,550, 1,600, 1,650, 1,700, 1,750, 1,800, 1,850, 1,900, 1,950, or 2,000fold higher than the corresponding wild-type protein.

In some embodiments, the present invention provides heme proteins,homologs, variants, and fragments thereof that catalyze enantioselectivecarbene insertion into silicon-hydrogen bonds with high enantiomericexcess. In particular embodiments, the heme proteins are variants orfragments thereof that catalyze enantioselective carbene insertion intosilicon-hydrogen bonds with higher enantiomeric excess values than thatof the corresponding wild-type protein. In some embodiments, the hemeprotein, homolog, variants, or fragment thereof catalyzes carbeneinsertion into silicon-hydrogen bonds with an enantiomeric excess valueof at least about 30% ee (e.g., at least about 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% ee). Preferably,the heme protein, homolog, variant, or fragment thereof catalyzescarbene insertion into silicon-hydrogen bonds with at least about 80% ee(e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ee). Morepreferably, the heme protein, homolog, variant, or fragment thereofcatalyzes carbene insertion into silicon-hydrogen bonds with at leastabout 95% ee (e.g., at least about 95%, 96%, 97%, 98%, 99%, or 100% ee).

B. Compounds

The methods of the invention can be used to provide a number oforganosilicon products. The organosilicon products include severalclasses of compound including, but not limited to, silicone compoundsand silicon-derived products (e.g., coatings, adhesives, and sealants),pharmaceutical compounds (i.e., drugs, therapeutic agents, etc.),biocompatible coatings, insecticides, and reagents for chemicalsynthesis. Examples of pharmaceutical compounds that can be preparedwith the methods of the invention include, but are not limited to,antiviral agents, antiparasitic agents, protease inhibitors (including,e.g., silanol enzyme inhibitors described by Sieburth in U.S. Pat. Nos.5,760,019 and 7,087,776), topoisomerase inhibitors (including, e.g.,camptothecin analogs described in U.S. Pat. No. 5,910,491 and WO98/07727), retinoids (including, e.g., bexarotene analogs described inWO 2004/048390), serotonin-norepinephrine reuptake inhibitors(including, e.g., venlafxine analogs described in WO 03/037905), andnon-steroidal anti-inflammatory drugs (e.g., indomethacin analogsdescribed in U.S. Pat. No. 7,964,738 and WO 2005/102358). Examples ofinsecticides that can be prepared with the methods of the inventioninclude, but are not limited to, silafluofen and pyrethroids disclosedin U.S. Pat. Nos. 4,709,068 and 4,883,789.

Examples of reagents for coating materials that can be prepared with themethods of the invention include, but are not limited to, silanes,siloxanes, and polysiloxanes disclosed in U.S. Pat. Nos. 9,359,386;8,952,118; 8,921,579; and U.S. Pat. No. 7,235,683.

Examples of reagents for chemical synthesis include, but are not limitedto, amines (e.g., 4-amino-3 ,3-dimethylbutylmethyldimethoxysilane,1-amino-2-(dimethylethoxysilyl) propane,N-(2-aminoethyl)-3-aminoisobutyldimethylmethoxysilane, and the like);amides (e.g., N-(trimethylsilyl)acetamide,N,O-bis(trimethylsilyl)acetamide, and the like); non-nantural aminoacids (e.g., trimethylsilylphenylalanine, trimethylsilylalanine,dimethylphenylsilylalanine, silaproline, and the like); acrylamides(e.g., 3-acrylamido-propyltrimethoxysilane,3-acrylamidopropyl-tris(trimethylsiloxy)silane, and the like); acrylates(e.g., acryloxymethyl-trimethoxysilane,(acryloxymethyl)phenethyltrimethoxy-silane, and the like); aromaticsilanes (e.g., bis(phenylethynyl)dimethylsilane,p-bromophenoxy-(t-butyl)dimethylsilane, N-benzyl-aminomethyltrim ethylsilane, benzyldimethylchlorosilane,N-benzyl-N-methoxymethyl-N-(tri-methylsilylmethyl)amine,(phenylaminomethyl)methyldimeth-oxysilane, and the like); poly-cyclicsilanes (e.g., [(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane,(5-bicyclo[2.2.1]hept-2-enyl)methyldiethoxysilane,(5-bicyclo[2.2.1]heptyl)dimethyl-chlorosilane,adamantylethyltri-chlorosilane, and the like); heteroaromatic silanes(e.g., bis(1-imidazolyl)dimethylsilane,3-(2-pyridylethyl)thiopropyltrimethoxysilane, 2-pyridyltrimethyl-silane,and the like); heterocyclic silanes (e.g.,2,2-bis(trimethylsilyl)-1,3-dithiane,bis(trimethyl-silyl)-5-fluorouracil, and the like); and phosphines(e.g., bis(trimethylsilyl)aminodimethyl-phosphine,diphenyl(trimethylsilyl-methyl)phosphine, and the like).

The organosilicon products can also serve as starting materials orintermediates for the synthesis of coatings, adhesives, sealants,pharmaceuticals, insecticides, chemical reagents, and other compounds.

In some embodiments, the methods of the present invention for producingorganosilicon products comprise combining a silicon-containing reagent,a carbene precursor, and a heme protein, homolog thereof, variantthereof, or fragment thereof as described herein under conditionssufficient to form an organosilicon product.

In some embodiments, the organosilicon product is a compound accordingto formula III:

For compounds of Formula III, R¹, R², R³, and R⁴ are independentlyselected from the group consisting of H, optionally substituted C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl,optionally substituted 6- to 10-membered heteroaryl, optionallysubstituted 6- to 10-membered heterocyclyl, cyano, halo, hydroxy,alkoxy, SR⁷, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, P(R⁹)₃, C(O)OR⁷, C(O)SR⁷,C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂ and C(O)NR⁷OR⁸. R⁵ and R⁶ areindependently selected from the group consisting of H, optionallysubstituted C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionallysubstituted C₆₋₁₀ aryl, optionally substituted 6- to 10-memberedheteroaryl, optionally substituted 6- to 10-membered heterocyclyl,cyano, halo, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂,C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷, and P(O)(OR7)₂. Each R⁷,R⁸, and R⁹ is independently selected from the group consisting of H,optionally substituted C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl.

In some embodiments, R¹, R², R³, and R⁴ in compounds of Formula III areindependently selected from the group consisting of H, optionallysubstituted C₁₋₁₈ alkyl, optionally substituted C₂₋₁₈ alkenyl,optionally substituted C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl; provided that at least one of R², R³, and R⁴ is other thanH; and R⁵ and R⁶ are independently selected from the group consisting ofH, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈haloalkyl, optionally substituted C₂₋₁₈ alkenyl, optionally substitutedC₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted6- to 10-membered heteroaryl, optionally substituted 6- to 10-memberedheterocyclyl, cyano, halo, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, P(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷, andP(O)(OR⁷)₂. In some such embodiments, each R⁷, R⁸, and R⁹ isindependently selected from the group consisting of H and optionallysubstituted C₁₋₆ alkyl.

In some embodiments, R¹ is H in compounds of Formula III; R², R³, and R⁴are independently selected from the group consisting of H, optionallysubstituted C₁₋₁₈ alkyl, optionally substituted C₂₋₁₈ alkenyl,optionally substituted C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl; provided that at least one of R², R³, and R⁴ is other thanH; and R⁵ and R⁶ are independently selected from the group consisting ofH, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈haloalkyl, optionally substituted C₂₋₁₈ alkenyl, optionally substitutedC₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted6- to 10-membered heteroaryl, optionally substituted 6- to 10-memberedheterocyclyl, cyano, halo, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, P(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷, andP(O)(OR⁷)₂. In some such embodiments, each R⁷, R⁸, and R⁹ isindependently selected from the group consisting of H and optionallysubstituted C₁₋₆ alkyl.

In some embodiments, R² is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl. In some embodiments, R³ and R⁴ are C₁₋₆ alkyl. In someembodiments, R⁵ is C(O)OR⁷. In some embodiments, R⁶ is selected from thegroup consisting of optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₁₋₁₈ haloalkyl, and optionally substituted C₂₋₁₈ alkenyl.

In general, the silicon-containing reagents useful in the methods of theinvention have the structure according to Formula I:

wherein R¹, R², R³, and R⁴ are as described above for compounds ofFormula III.

In some embodiments, R¹, R², R³, and R⁴ are independently selected fromthe group consisting of H, optionally substituted C₁₋₁₈ alkyl,optionally substituted C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted C₆₋₁₀aryl-C₁₋₆ alkyl, optionally substituted 6- to 10-membered heteroaryl,and optionally substituted 6- to 10-membered heterocyclyl.

In some embodiments, R¹ is H; and R², R³, and R⁴ are independentlyselected from the group consisting of H, optionally substituted C₁₋₁₈alkyl, optionally substituted C₂₋₁₈ alkenyl, optionally substitutedC₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substitutedC₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6- to 10-memberedheteroaryl, and optionally substituted 6- to 10-membered heterocyclyl;provided that at least one of R², R³, and R⁴ is other than H.

In some embodiments, R¹ is selected from the group consisting of B(R⁹)₂and Si(R⁹)₃; and R², R³, and R⁴ are independently selected from thegroup consisting of H, optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted C₆₋₁₀aryl-C₁₋₆ alkyl, optionally substituted 6- to 10-membered heteroaryl,and optionally substituted 6- to 10-membered heterocyclyl; provided thatat least one of R², R³, and R⁴ is other than H.

In some embodiments, R², R³, and R⁴ are independently selected from thegroup consisting of H, optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₂₋₁₈ alkenyl, and optionally substituted C₂₋₁₈ alkynyl. Insome embodiments, R², R³, and R⁴ are independently selected from thegroup consisting of optionally substituted C₆₋₁₀ aryl, optionallysubstituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6- to10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl.

In some embodiments, R² is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl. In some embodiments, R³ and R⁴ are C₁₋₆ alkyl. One or bothof R³ and R⁴ can be for, example, methyl, ethyl, n-propyl, isopropyl,n-butyl, or t-butyl. In some embodiments, R³ and R⁴ are C₁₋₄ alkyl. Insome embodiments, R³ and R⁴ are methyl.

In some embodiments, R² is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl; and R³ and R⁴ are independently selected C₁₋₆ alkyl.

In some embodiments, R² is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl; and R³ and R⁴ are methyl. In some embodiments, R² isselected from the group consisting of optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl; and R³ and R⁴ are methyl. In some embodiments, R² isoptionally substituted phenyl; and R³ and R⁴ are independently selectedC₁₋₆ alkyl. In some embodiments, R² is optionally substituted phenyl;and R³ and R⁴ are methyl.

In some embodiments, R² is phenyl, which is optionally substituted withalkyl, alkenyl, alkynyl, halo, hydroxy, amino, alkylamino, alkoxy,haloalkyl, carboxy, alkyl carboxylate, amido, nitro, oxo, or cyano. Insome embodiments, R² is phenyl, which is optionally substituted withalkyl, alkenyl, alkynyl, haloalkyl, halo, hydroxy, amino, alkylamino,alkoxy, haloalkyl, carboxy, alkyl carboxylate, amido, nitro, oxo, orcyano.

In some embodiments, the silicon-containing reagent has a structureaccording to Formula Ia:

wherein R^(2a) is selected from the group consisting of H, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, hydroxy, C₁₋₆ alkoxy, halo, C₁₋₆ haloalkyl,C₁₋₄ alkyl carboxylate, amino, and C₃₋₆ alkylamino.

In some embodiments, the silicon-containing reagent has a structureaccording to Formula Ib:

wherein R^(2a) is selected from the group consisting of H, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, hydroxy, C₁₋₆ alkoxy, halo, C₁₋₆ haloalkyl,C₁₋₄ alkyl carboxylate, amino, and C₃₋₆ alkylamino.

In some embodiments, the silicon-containing reagent has a structureaccording to Formula I, wherein R² is selected from the group consistingof benzyl, naphthyl, 3,4-dihydro-2H-pyran-2-yl, benzofuran-2-yl, andbenzo[b]thiophen-2-yl.

A number of carbene precusors can be used in the methods and reactionmixtures of the invention including, but not limited to, amines, azides,hydrazines, hydrazones, epoxides, diazirenes, and diazo reagents. Insome embodiments, the carbene precursor is an epoxide (i.e., a compoundcontaining an epoxide moiety). The term “epoxide moiety” refers to athree-membered heterocycle having two carbon atoms and one oxygen atomconnected by single bonds. In some embodiments, the carbene precursor isa diazirene (i.e., a compound containing a diazirine moiety). The term“diazirine moiety” refers to a three-membered heterocycle having onecarbon atom and two nitrogen atoms, wherein the nitrogen atoms areconnected via a double bond. Diazirenes are chemically inert, smallhydrophobic carbene precursors described, for example, in US2009/0211893, by Turro (J. Am. Chem. Soc. 1987, 109, 2101-2107), and byBrunner (J. Biol. Chem. 1980, 255, 3313-3318), which are incorporatedherein by reference in their entirety.

In some embodiments, the carbene precursor is a diazo reagent. In someembodiments, the diazo reagent has a structure according to Formula II:

wherein R⁵ and R⁶, as well as R⁷, R⁸, and R⁹ within R⁵ and R⁶, are asdescribed above for compounds of Formula III.

In some embodiments, R⁵ and R⁶ are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₁₋₁₈ haloalkyl, optionally substituted C₂₋₁₈ alkenyl,optionally substituted C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl,optionally substituted 6- to 10-membered heteroaryl, optionallysubstituted 6- to 10-membered heterocyclyl, cyano, halo, N(R⁸)₂, B(R⁹)₂,Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸,C(O)C(O)OR⁷, and P(O)(OR⁷)₂. Each R⁷, R⁸, and R⁹ is independentlyselected from the group consisting of H and optionally substituted C₁₋₆alkyl. One, two, or three of R⁷, R⁸, and R⁹ can be, for example, methyl,ethyl, n-propyl, isopropyl, n-butyl, or t-butyl. In some embodiments,one, two, or three of R⁷, R⁸, and R⁹ are H.

In some embodiments, R⁵ and R⁶ are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₁₋₁₈ haloalkyl, optionally substituted C₂₋₁₈ alkenyl,optionally substituted C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl,optionally substituted 6- to 10-membered heteroaryl, optionallysubstituted 6- to 10-membered heterocyclyl, cyano, and halo. In someembodiments, R⁵ and R⁶ are independently selected from the groupconsisting of N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, and P(O)(OR⁷)₂. In someembodiments, R⁵ and R⁶ are independently selected from the groupconsisting of C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂,C(O)NR⁷OR⁸, and C(O)C(O)OR⁷.

In some embodiments, R⁵ is C(O)OR⁷. In some embodiments, R⁶ is selectedfrom the group consisting of optionally substituted C₁₋₁₈ alkyl,optionally substituted C₆₋₁₀ aryl, optionally substituted C₁₋₁₈haloalkyl, and optionally substituted C₂₋₁₈ alkenyl.

In some embodiments, R⁵ is C(O)OR⁷ and R⁷ is optionally substituted C₁₋₆alkyl. In some embodiments, R⁵ is C(O)OR⁷ and R⁷ is selected from thegroup consisting of methyl and ethyl. In some embodiments, R⁵ isC(O)OR⁷; R⁷ is selected from the group consisting of methyl and ethyl;and R⁶ is selected from the group consisting of C₁₋₁₂ alkyl, C₆₋₁₀ aryl,C₁₋₁₂ haloalkyl, and C₂₋₁₂ alkenyl. In some embodiments, R⁵ is C(O)OR⁷and R⁷ is ethyl. In some embodiments, R⁵ is C(O)R⁷; R⁷ is ethyl; and R⁶is selected from the group consisting of methyl, ethyl, phenyl,trifluoromethyl, and allyl (i.e., CH₃—CH═CH—).

In some embodiments, the diazo reagent is selected from an α-diazoester,an α-diazoamide, an α-diazonitrile, an α-diazoketone, anα-diazoaldehyde, and an α-diazosilane. In some embodiments, the diazoreagent has a formula selected from:

wherein each R⁷ and R⁹ is independently selected from H, optionallysubstituted C₁₋₁₂ alkyl, optionally substituted C₂₋₁₂ alkenyl, andoptionally substituted C₆₋₁₀ aryl.

Diazo reagents can be formed from a number of starting materials usingprocedures that are known to those of skill in the art. Ketones(including 1,3-diketones), esters (including β-ketones), acyl chlorides,and carboxylic acids can be converted to diazo reagents employing diazotransfer conditions with a suitable transfer reagent (e.g., aromatic andaliphatic sulfonyl azides, such as toluenesulfonyl azide,4-carboxyphenylsulfonyl azide, 2-naphthalenesulfonyl azide,methylsulfonyl azide, and the like) and a suitable base (e.g.,triethylamine, triisopropylamine, diazobicyclo[2.2.2]octane,1,8-diazabicyclo[5.4.0]undec-7-ene, and the like) as described, forexample, in U.S. Pat. No. 5,191,069 and by Davies (J. Am. Chem. Soc.1993, 115, 9468-9479), which are incorporated herein by reference inthere entirety. The preparation of diazo compounds from azide andhydrazone precursors is described, for example, in U.S. Pat. Nos.8,350,014 and 8,530,212, which are incorporated herein by reference inthere entirety. Alkylnitrite reagents (e.g., (3-methylbutyl)nitrite) canbe used to convert α-aminoesters to the corresponding diazocompounds innon-aqueous media as described, for example, by Takamura (Tetrahedron,1975, 31: 227), which is incorporated herein by reference in itsentirety. Alternatively, a diazo compound can be formed from analiphatic amine, an aniline or other arylamine, or a hydrazine using anitrosating agent (e.g., sodium nitrite) and an acid (e.g.,p-toluenesulfonic acid) as described, for example, by Zollinger (DiazoChemistry I and II, VCH Weinheim, 1994) and in US 2005/0266579, whichare incorporated herein by reference in their entirety.

In some embodiments, the invention provides methods and reactionmixtures for producing an organosilicon product wherein the carbeneprecursor is an amine. In some embodiments, the amine is converted to adiazo reagent by contacting the amine with a nitrosating agent underconditions sufficient to form the diazo reagent. In some embodiments,the nitrosating agent is selected from the group consisting of sodiumnitrite, potassium nitrite, lithium nitrite, calcium nitrite, magnesiumnitrite, ethyl nitrite, n-butyl nitrite, and (3-methylbutyl)nitrite. Inthe some embodiments, the amine is contacted with the nitrosating agentin the presence of an acid. Examples of suitable acids include, but arenot limited to, sulfonic acids (e.g., p-toluenesulfonic acid,methanesulfonic acid, and the like), phosphoric acid, nitric acid,sulfuric acid, and hydrochloric acid. In some embodiments, thenitrosating agent is sodium nitrite. In some embodiments, thenitrosating agent is sodium nitrite and the acid is p-toluenesulfonicacid. In some embodiments, the nitrosating agent is sodium nitrite, theacid is p-toluenesulfonic acid, and the amine is selected from the groupconsisting of an alkylamine, an arylamine, an α-aminoketone, anα-aminoester, and an α-aminoamide. In some embodiments, the amine iscontacted with the nitrosating reagent (and the acid, when used) in asuitable organic solvent (e.g., acetonitrile, N,N-dimethylformamide,dimethylsulfoxide, and the like) for a time sufficient to form the diazoreagent prior to combination with the silicon-containing reagent and theheme protein used for forming the organosilicon product. The diazoreagent in the organic solvent can then be combined with a mixturecontaining the silicon-containing reagent and the heme protein. In someembodiments, the mixture containing the silicon-containing reagent andthe heme protein is an aqueous mixture containing a suitable buffer asdescribed below. Alternatively, the amine can be converted to the diazoreagent in situ, by combining the amine, the nitrosating agent, and theacid (when used) directly with the the silicon-containing reagent andthe heme protein.

C. Reaction Conditions

The methods of the invention include forming reaction mixtures thatcomprise a silicon-containing reagent, a carbene precursor, and a hemeprotein, fragment thereof, homolog thereof, or variant thereof asdescribed above.

The heme proteins, fragments thereof, homologs thereof, or variantsthereof can be, for example, purified prior to addition to a reactionmixture or secreted by a cell present in the reaction mixture. Thereaction mixture can contain a cell lysate including the heme protein,fragment thereof, homolog thereof, or variant thereof, as well as otherproteins and other cellular materials. Alternatively, a heme protein,fragment thereof, homolog thereof, or variant thereof can catalyze thereaction within a cell expressing the heme protein, fragment thereof,homolog thereof, or variant thereof. Any suitable amount of hemeprotein, fragment thereof, homolog thereof, or variant thereof can beused in the methods of the invention. In general, silicon-hydrogencarbene insertion reaction mixtures contain from about 0.01 mol % toabout 10 mol % heme protein with respect to the carbene precursor (e.g.,diazo reagent) and/or silicon-containing reagent. The reaction mixturescan contain, for example, from about 0.01 mol % to about 0.1 mol % hemeprotein, or from about 0.1 mol % to about 1 mol % heme protein, or fromabout 1 mol % to about 10 mol % heme protein. The reaction mixtures cancontain from about 0.05 mol % to about 5 mol % heme protein, or fromabout 0.05 mol % to about 0.5 mol % heme protein. The reaction mixturescan contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or about1 mol % heme protein.

One of skill in the art will appreciate that depending on the structureof the carbene precursor and/or silicon-containing reagent,silicon-hydrogen carbene insertion reaction mixtures of the presentinvention can comprise a carbene precursor, a silicon-containingreagent, and an isolated heme group. The heme group can comprise aporphyrin molecule and an iron cofactor. In particular embodiments, theporphyrin molecule contains a non-native cofactor (i.e., a metal otherthan iron), non-limiting examples of which include cobalt, rhodium,copper ruthenium, iridium, and manganese. The cofactor can be any metal,as long as it catalyzes carbon-silicon bond formation. The heme groupcan catalyze carbon-silcon bond formation in vitro, or can be present ina cell and the carbon-silicon bond formation can occur in vivo. For invivo reactions, any suitable host cell described herein can be used. Thereaction mixtures can contain, for example, from about 0.01 mol % toabout 0.1 mol % heme, or from about 0.1 mol % to about 1 mol % heme, orfrom about 1 mol % to about 10 mol % heme. The reaction mixtures cancontain from about 0.05 mol % to about 5 mol % heme, or from about 0.05mol % to about 0.5 mol % heme. The reaction mixtures can contain about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or about 1 mol % heme.

The concentration of silicon-containing reagent and carbene precursor(e.g., diazo reagent) are typically in the range of from about 100 μM toabout 1 M. The concentration can be, for example, from about 100 μM toabout 1 mM, or about from 1 mM to about 100 mM, or from about 100 mM toabout 500 mM, or from about 500 mM to 1 M. The concentration can be fromabout 500 μM to about 500 mM, 500 μM to about 50 mM, or from about 1 mMto about 50 mM, or from about 15 mM to about 45 mM, or from about 15 mMto about 30 mM. The concentration of silicon-containing reagent orcarbene precursor can be, for example, about 100, 200, 300, 400, 500,600, 700, 800, or 900 μM. The concentration of silicon-containingreagent or carbene precursor can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,150, 200, 250, 300, 350, 400, 450, or 500 mM.

Reaction mixtures can contain additional reagents. As non-limitingexamples, the reaction mixtures can contain buffers (e.g., M9-N buffer,2-(N-morpholino)ethanesulfonic acid (MES),2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES),3-morpholinopropane-1-sulfonic acid (MOPS),2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate,sodium phosphate, phosphate-buffered saline, sodium citrate, sodiumacetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide,dimethylformamide, ethanol, methanol, isopropanol, glycerol,tetrahydrofuran, acetone, acetonitrile, and acetic acid), salts (e.g.,NaCl, KCl, CaCl₂, and salts of Mn²⁺ and Mg²⁺), denaturants (e.g., ureaand guandinium hydrochloride), detergents (e.g., sodium dodecylsulfateand Triton-X 100), chelators (e.g., ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),2-({2-[Bis(carboxymethyl)amino]ethyl} (carboxymethyl)amino)acetic acid(EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid(BAPTA)), sugars (e.g., glucose, sucrose, and the like), and reducingagents (e.g., sodium dithionite, NADPH, dithiothreitol (DTT),β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)).Buffers, cosolvents, salts, denaturants, detergents, chelators, sugars,and reducing agents can be used at any suitable concentration, which canbe readily determined by one of skill in the art. In general, buffers,cosolvents, salts, denaturants, detergents, chelators, sugars, andreducing agents, if present, are included in reaction mixtures atconcentrations ranging from about 1 μM to about 1 M. For example, abuffer, a cosolvent, a salt, a denaturant, a detergent, a chelator, asugar, or a reducing agent can be included in a reaction mixture at aconcentration of about 1 μM, or about 10 μM, or about 100 μM, or about 1mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 100 mM, orabout 250 mM, or about 500 mM, or about 1 M. In some embodiments, areducing agent is used in a sub-stoichiometric amount with respect tothe olefin substrate and the diazo reagent. Cosolvents, in particular,can be included in the reaction mixtures in amounts ranging from about1% v/v to about 75% v/v, or higher. A cosolvent can be included in thereaction mixture, for example, in an amount of about 5, 10, 20, 30, 40,or 50% (v/v).

Reactions are conducted under conditions sufficient to catalyze theformation of an organosilicon product. The reactions can be conducted atany suitable temperature. In general, the reactions are conducted at atemperature of from about 4° C. to about 40° C. The reactions can beconducted, for example, at about 25° C. or about 37° C. The hemeproteins or cells expressing or containing the heme proteins can be heattreated. In some embodiments, heat treatment occurs at a temperature ofabout 75° C. The reactions can be conducted at any suitable pH. Ingeneral, the reactions are conducted at a pH of from about 6 to about10. The reactions can be conducted, for example, at a pH of from about6.5 to about 9 (e.g., about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, or 9.0). The reactions can be conducted for any suitablelength of time. In general, the reaction mixtures are incubated undersuitable conditions for anywhere between about 1 minute and severalhours. The reactions can be conducted, for example, for about 1 minute,or about 5 minutes, or about 10 minutes, or about 30 minutes, or about 1hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 12hours, or about 24 hours, or about 48 hours, or about 72 hours. Thereactions can be conducted for about 1 to 4 hours (e.g., 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4 hours).Reactions can be conducted under aerobic conditions or anaerobicconditions. Reactions can be conducted under an inert atmosphere, suchas a nitrogen atmosphere or argon atmosphere. In some embodiments, asolvent is added to the reaction mixture. In some embodiments, thesolvent forms a second phase, and the carbene insertion intosilicon-hydrogen bonds occurs in the aqueous phase. In some embodiments,the heme protein, fragment thereof, variant thereof, or homolog thereof,is located in the aqueous layer whereas the substrates and/or productsoccur in an organic layer. Other reaction conditions may be employed inthe methods of the invention, depending on the identity of a particularheme protein, silicon-containing reagent, or carbene precursor (e.g.,diazo reagent).

Reactions can be conducted in vivo with intact cells expressing a hemeenzyme of the invention. The in vivo reactions can be conducted with anyof the host cells used for expression of the heme enzymes, as describedherein. A suspension of cells can be formed in a suitable mediumsupplemented with nutrients (such as mineral micronutrients, glucose andother fuel sources, and the like). Organosilicon product yields fromreactions in vivo can be controlled, in part, by controlling the celldensity in the reaction mixtures. Cellular suspensions exhibitingoptical densities ranging from about 0.1 to about 50 at 600 nm can beused for silicon-hydrogen carbene insertion reactions. Other densitiescan be useful, depending on the cell type, specific heme proteins, orother factors.

The methods of the invention can be assessed in terms of thediastereoselectivity and/or enantioselectivity of carbene insertion intosilicon—hydrogen bonds—that is, the extent to which the reactionproduces a particular isomer, whether a diastereomer or enantiomer. Aperfectly selective reaction produces a single isomer, such that theisomer constitutes 100% of the product. As another non-limiting example,a reaction producing a particular enantiomer constituting 90% of thetotal product can be said to be 90% enantioselective. A reactionproducing a particular diastereomer constituting 30% of the totalproduct, meanwhile, can be said to be 30% diastereoselective.

In general, the methods of the invention include reactions that are fromabout 1% to about 99% diastereoselective. The reactions are from about1% to about 99% enantioselective. The reaction can be, for example, fromabout 10% to about 90% diastereoselective, or from about 20% to about80% diastereoselective, or from about 40% to about 60%diastereoselective, or from about 1% to about 25% diastereoselective, orfrom about 25% to about 50% diastereoselective, or from about 50% toabout 75% diastereoselective. The reaction can be about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, orabout 95% diastereoselective. The reaction can be from about 10% toabout 90% enantioselective, from about 20% to about 80%enantioselective, or from about 40% to about 60% enantioselective, orfrom about 1% to about 25% enantioselective, or from about 25% to about50% enantioselective, or from about 50% to about 75% enantioselective.The reaction can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95% enantioselective.Accordingly some embodiments of the invention provide methods whereinthe reaction is at least 30% to at least 90% diastereoselective. In someembodiments, the reaction is at least 30% to at least 90%enantioselective. Preferably, the reaction is at least 80% (e.g., atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) enantioselective. Morepreferably, the reaction is at least 90% (e.g., at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) enantioselective.

IV. Examples

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes only, and are not intended to limit the invention in anymanner. Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1. Enzymatic Carbon-Silicon Bond Formation to AffordOrganosilicon Compounds Using Cytochrome P450 Proteins and Other HemeProteins

This example shows the production of organosilicon compounds usingvarious heme proteins, including cytochrome P450 proteins.

P450 Expression and Purification

One liter Hyperbroth (0.1 mg/mL ampicillin) was inoculated with anovernight culture (25 mL LB, 0.1 mg/mL ampicillin) of recombinant E.coli BL21 cells harboring a pCWori or pET22 plasmid encoding cytochromeP450 variants under the control of the tac or T7 promoter. The cultureswere shaken at 200 rpm at 37° C. for roughly 3.5 hours, or until anoptical of density of 1.2-1.8 was reached. The temperature was reducedto 20° C. and the shake rate was reduced to 130-150 rpm for 20 minutes,then the cultures were induced by adding isopropylβ-D-1-thiogalactopyranoside (IPTG) and aminolevulinic acid to a finalconcentration of 0.25 mM and 0.5 mM, respectively. The cultures wereallowed to continue for another 20 hours at this temperature and shakerate. Cell were harvested by centrifugation (4° C., 15 minutes,3,000×g), and the cell pellet was stored at −20° C. or below for atleast 2 hours. For the purification of 6XHis tagged cytochrome P450s,the thawed cell pellet was resuspended in Ni-NTA buffer A (25 mMTris.HC1, 200 mM NaCl, 25 mM imidazole, pH 8.0, 4 mL/gcw) and lysed bysonication (2×1 min, output control 5, 50% duty cycle). The lysate wascentrifuged at 27,000×g for 20 minutes at 4° C. to remove cell debris.The collected supernatant was first subjected to a Ni-NTA chromatographystep using a Ni Sepharose column (HisTrap-HP, GE healthcare, Piscataway,N.J.). The cytochrome P450 was eluted from the Ni Sepharose column using25 mM Tris.HCl, 200 mM NaCl, 300 mM imidazole, pH 8.0. Ni-purifiedprotein was buffer exchanged into 0.1 M phosphate buffer (pH 8.0) usinga 30 kDa molecular weight cut-off centrifugal filter. Proteinconcentrations were determined by CO-assay. For storage, proteins wereportioned into 300 μL aliquots and stored at −80° C.

Small-Scale Carbon—Silicon Bond Forming Reactions in vitro and in vivoUnder Anaerobic Conditions

The in vitro and in vivo carbon-silicon bond forming reaction procedurescatalyzed by cytochrome P450s and other heme proteins were identical tothose described in Example 2 for Rma cyt c. The results are presentedFIG. 1 and demonstrate that a variety of heme proteins (myoglobin, P450,globin and cytochrome c) functioned as carbon-silicon bond formingcatalysts to give product of Formula III.

Small-Scale Carbon—Silicon Bond Forming Reactions Under AerobicConditions

The procedures for carbon-silicon bond formation under aerobicconditions were similar to those described for anaerobic conditions,except that the reactions were carried out under air.

Example 2. Enzymatic Carbon—Silicon Bond Formation to AffordOrganosilicon Compounds Using Rma cyt c Variants in vitro and in vivo

This example shows the use of cytochrome c protein from Rhodothermusmarinus (Rma cyt c) to catalyze the formation of organosiliconcompounds.

Cytochrome c Expression

One liter Hyperbroth (100 μg/mL ampicillin, 20 μg/mL chloramphenicol)was inoculated with an overnight culture of 20 mL LB (100 μg/mLampicillin, 20 μg/mL chloramphenicol). The overnight culture containedrecombinant E. coli BL21-DE3 cells harboring a pET22 plasmid and pEC86plasmid (Arslan et al. Biochem. Biophys. Res. Commun. 251, 744-747(1998)) encoding the cytochrome c variant under the control of the T7promoter, and the cytochrome c maturation (ccm) operon under the controlof a tet promoter, respectively. The cultures were shaken at 200 rpm at37° C. for approximately 2 hours or until an optical of density of0.6-0.9 was reached. The flask containing the cells was placed on icefor 30 minutes. The incubator temperature was reduced to 20° C.,maintaining the 200 rpm shake rate. Cultures were induced by adding IPTGand aminolevulinic acid to a final concentration of 20 μM and 200 μM,respectively. The cultures were allowed to continue for another 20-24hours at this temperature and shake rate. Cells were harvested bycentrifugation (4° C., 15 minutes, 3,000×g) to produce a cell pellet.

Preparation of Whole Cell and Heat-Treated Lysate Catalysts

To prepare whole cells for catalysis, the cell pellet prepared in theprevious paragraph was resuspended in M9-N minimal media (M9 mediawithout ammonium chloride) to an optical density (OD600) of 60. Toprepare heat-treated lysate for catalysis, whole cells in M9-N minimalmedia at OD600=15 were placed in a water bath at 75° C. for 10 minutes.After the time at 75° C., the sample was centrifuged to remove theprecipitate (4° C., 10 minutes, 4,000×g). The supernatant was collectedand used as the heat-treated lysate catalyst, while the pellet wasdiscarded.

Purification of Rma cyt c

To prepare purified proteins, the cell pellet prepared as describedabove was stored at −20° C. or below overnight. For the purification of6XHis tagged cytochrome c proteins, the thawed cell pellet wasresuspended in Ni-NTA buffer A (25 mM Tris.HCl, 200 mM NaCl, 25 mMimidazole, pH 8.0, 4 mL/gcw) and lysed by sonication (2 minutes,2-second pulse, 2 seconds off, 50% amplitude on a Q500 Qsonicasonicator). After sonication, the sample was centrifuged at 27,000×g for10 minutes at 4° C. to remove cell debris. The sample was then placed ina water bath at 75° C. for 10 minutes, and centrifuged at 27,000×g for10 minutes at 4° C. to remove cell debris. The collected supernatant waspurified using an AKTA purifier 10 FPLC with a Ni Sepharose column(HisTrap-HP, GE healthcare, Piscataway, N.J.). The cytochrome c waseluted from the Ni Sepharose column using 25 mM Tris.HCl, 200 mM NaCl,300 mM imidazole, pH 8.0. The Ni-purified protein was buffer exchangedinto 0.1 M phosphate buffer (pH 8.0) using a 10 kDa molecular weightcut-off centrifugal filter, then dialyzed overnight at 4° C. in 20 mMphosphate buffer (pH 8.0) using a 3.5 kDa molecular weight cut-offdialysis bag. Protein concentrations were determined by BCA assay, usingBSA to create the standard curve. For storage, proteins were portionedinto 300 μL aliquots and stored at −80° C.

Small-Scale Carbon—Silicon Bond Forming Reactions in Heat-Treated LysateUnder Anaerobic Conditions

Small-scale (400 μL) reactions were carried out in 2 mL glass crimpvials (Agilent Technologies, San Diego, Calif.). Heat-treated lysate(340 μL) was added to an unsealed crimp vial before crimp sealing with asilicone septum. The headspace of the vial was flushed with argon for 10minutes (no bubbling). A solution of sodium dithionite (40 μL, 100 mM)was added, followed by a solution of silicon-containing reagent ofFormula I (10 μL, 400 mM in MeCN; for example, PhMe2SiH) and a solutionof diazo reagent of Formula II (10 μL, 400 mM in MeCN; for example,ethyl 2-diazopropanoate or Me-EDA). The reaction vial was left to shakeon a plate shaker at 400 rpm for 1.5 hours at room temperature. Toquench the reaction, the vial was uncapped and cyclohexane (1 mL) wasadded, followed by 2-phenylethanol (20 μL, 20 mM in cyclohexane) as aninternal standard. The mixture was transferred to a 1.5 mL Eppendorftube and vortexed and centrifuged (14000×ref, 5 minutes). The organiclayer was analyzed by gas chromatography (GC) and supercritical fluidchromatography (SFC). The results of the small scale reactions arepresented in FIG. 2 and demonstrate that Rma cyt c and variants thereofwere capable of catalyzing the formation of carbon-silicon bonds to giveproduct of formula III with high turnover number and selectivity.Specifically, the best variant found in the initial screen of Rma cyt cvariants comprised the mutations V75T, M100D, and M103E, which catalyzedthe desired reaction with greater than 1,800 total turnover number (TTN)and greater than 99% ee.

Small-Scale Whole Cells Catalysis of Carbon—Silicon Bond Formation

Small-scale (400 μL) reactions were carried out in 2 mL glass crimpvials (Agilent Technologies, San Diego, Calif.). Whole cell catalysts(340 μL, OD600=60 in M9-N minimal media) were added to an unsealed crimpvial before crimp sealing with a silicone septum. The headspace of thevial was flushed with argon for 10 minutes (no bubbling). A solution ofglucose (40 μL, 250 mM) was added, followed by a solution of siliconreagent of Formula I (10 μL, 400 mM in MeCN; for example, PhMe2SiH) anda solution of diazo reagent of Formula II (10 μL, 400 mM in MeCN; forexample, ethyl 2-diazopropanoate or Me-EDA). The reaction vial was leftto shake on a plate shaker at 400 rpm for 1.5 hours at room temperature.To quench the reaction, the vial was uncapped and cyclohexane (1 mL) wasadded, followed by 2-phenylethanol (20 μL, 20 mM in cyclohexane) as aninternal standard. The mixture was transferred to a 1.5 mL Eppendorftube and vortexed and centrifuged (14000×ref, 5 minutes). The organiclayer was analyzed by gas chromatography (GC) and supercritical fluidchromatography (SFC).

The results of the small scale reactions are presented in FIG. 3 anddemonstrate that Rma cyt c and variants thereof functioned as whole-cellcatalysts and promoted the formation of carbon-silicon bonds to giveproducts of Formula III with high selectivity. Specifically, the bestvariant found in the initial screen of Rma cyt c variants comprised themutations V75T, M100D and M103E, which provided product of formula IIIin greater than 99% ee.

Example 3. Directed Evolution of Cytochrome c for Carbon-Silicon BondFormation

This example shows the generation of mutant variants of Rhodothermusmarinus (Rma) cytochrome c and demonstrates their ability to act ascarbon-silicon bond-forming catalysts in vitro and in vivo.

Introduction

Silicon constitutes almost 30% of the mass of the Earth's crust, yet nolife form is known to have the ability to forge carbon—silicon bonds(1). Despite the absence of organosilicon compounds in the biologicalworld, synthetic chemistry has enabled the appreciation of the uniqueand desirable properties that have led to their broad applications inchemistry and materials science (2,3). As a biocompatible carbonisostere, silicon can also be used to optimize and repurpose thepharmaceutical properties of bioactive molecules (4,5).

The natural supply of silicon may be abundant, but sustainable methodsfor synthesizing organosilicon compounds are not (6-8). Carbon—siliconbond forming methods that introduce silicon motifs to organic moleculesenantioselectively rely on multi-step synthetic campaigns to prepare andoptimize chiral reagents or catalysts; precious metals are alsosometimes needed to achieve the desired activity (9-15). Syntheticmethodologies such as carbene insertion into silanes can be renderedenantioselective using chiral transition metal complexes based onrhodium (11,12), iridium (13) and copper (14,15). These catalysts canprovide optically pure products, but not without limitations: theyrequire halogenated solvents and sometimes low temperatures to functionoptimally and have limited turnovers (less than 100). Examples of knowncatalytic systems are listed in Tables 2-4. α-Alkyl diazo compounds arechallenging substrates for intermolecular carbene-transfer chemistry dueto their propensity to undergo competing intramolecular β-hydridemigration (120,85). As a result, only a subset of catalytic systemsshown in Table 2 have been reported to accommodate these substrates, assummarized in Table 3.

TABLE 2 Summary of known catalytic systems for enantioselective carbeneinsertion into silicon- hydrogen bonds Chiral catalytic Substrate systemRef Reaction condition scope TTN % ee Copper A* (87) CH₂Cl₂, rt, 18 h 417 to 25 17 to 98 B (108) C₆H₆, 0° C., 13.5 h 1  8 to 10 29 to 78 C*(86) CH₂Cl₂, −60 to 0° C., 2-12 h 24  3 to 19 12 to 99 B (109) CH₂Cl₂,−40 to 0° C., 48-72 h 8 5 to 9 49 to 88 B (110) CH₂Cl₂, −40 to −10° C. 95 to 8 40 to 84 Iridium D* (85) CH₂Cl₂, −78 or −30° C., 24 h 15 24 to 5094 to 99 E (111) CH₂Cl₂, −78° C., 24 h 7 75 to 94 72 to 91 Rhodium F*(84) CH₂Cl₂, rt to 40° C., 6-12 h 34  9 to 30 77 to 99 G (112) CH₂Cl₂,−78° C. or rt, 3-12 h 5  8 to 45 20 to 63 H (108) C₅H₁₂, −78 to −75° C.,24 h 1 24 to 70 48 to 97 I* (83) CF₃CH₂OH, −35° C. 10 51 to 97 20 to 99H (113) CH₂Cl₂, −78° C. then rt, 23 h 6 22 to 54 77 to 94 variousRh(II)- (114) CH₂Cl₂, rt or −78° C., 0.5 to 6 <1 to 23  6 to 76carboxylate (115) 24 h to overnight J (116) CH₂Cl₂, −78° C. to rt, 23 h2 35 to 40 38 to 58 H (117) CH₂Cl₂, −78° C., 1 to 1.5 h 4 34 to 43 35 to72 H (118) C₅H₁₂, −78° C., 24 h 5 13 to 19 75 to 95 various Rh(II)-(119) CH₂Cl₂, rt or reflux 1  8 to 17  6 to 47 carboxylate Catalyticsystems that could afford enantiopure products are denoted with (*). rt= room temperature. Chemical structures of catalysts are shown in FIG.4.

TABLE 3 Summary of known catalytic systems that can accept α-alkyl diazocompounds as substrates for enantioselective carbene insertion intosilicon-hydrogen bonds Chiral catalytic Substrate system Ref Reactioncondition scope TTN % ee Copper A (87) CH₂Cl₂, rt, 18 h 4 17 to 25 17 to98 C (86) CH₂Cl₂, −40° C., 2-12 h 2  3 to 12 12 to 35 Iridium D (85)CH₂Cl₂, −78° C., 24 h 8 24 to 44 94 to 99 Rhodium F (84) CH₂Cl₂, rt to40° C., 6-12 h 2 11 to 15 70 to 77 I (83) CF₃CH₂OH, −35° C. 1 N/A 64 H(113) CH₂Cl₂, −78° C. then rt, 23 h 5 22 to 54 77 to 94Chemical structures of chiral catalysts are shown in FIG. 4. rt denotesroom temperature.

TABLE 4 Summary of known catalytic systems for reaction betweenphenyldimethylsilane and Me-EDA via enantioselective carbene insertioninto silicon-hydrogen bonds

Chiral catalytic system Ref Reaction condition TTN % ee Copper C (86)CH₂Cl₂, −40° C., 2-12 h 12 35 Iridium D (85) CH₂Cl₂, −78° C., 24 h 43 97Rhodium F (84) CH₂Cl₂, rt to 40° C., 6-12 h 15 77Chemical structures of chiral catalysts are shown in FIG. 4. rt denotesroom temperature.

Because of their ability to accelerate chemical transformations withexquisite specificity and selectivity, enzymes are increasingly soughtafter complements to or even replacements for chemical synthesis methods(16,17). Biocatalysts that are fully genetically encoded and assembledinside of cells are readily tunable using molecular biology techniques.They can be produced at low cost from renewable resources in microbialsystems and perform catalysis under mild conditions. Although naturedoes not use enzymes to form carbon—silicon bonds, the proteinmachineries of living systems are often “promiscuous,” that is, capableof catalyzing reactions distinct from their biological functions.Evolution, natural or in the laboratory, can use these promiscuousfunctions to generate catalytic novelty (18-20). For example, hemeproteins can catalyze a variety of non-natural carbene transferreactions in aqueous media, including N—H and S—H insertions, which canbe greatly enhanced and made exquisitely selective by directed evolution(21-23).

Results and Discussion

Based on the idea that heme proteins can also catalyze carbene insertioninto silicon—hydrogen bonds, directed evolution was used to createenzymes of the present invention. Because iron is not known to catalyzethis transformation (24), it was first examined whether free heme couldfunction as a catalyst in aqueous media. Initial experiments showed thatthe reaction between phenyldimethylsilane and ethyl 2-diazopropanoate(Me-EDA) in neutral buffer (M9-N minimal medium, pH 7.4) at roomtemperature gave racemic organosilicon (compound 3) at very low levels,a total turnover number (TTN) of 4 (FIG. 5A). No product formation wasobserved in the absence of heme, and the organosilicon product wasstable under the reaction conditions.

Next, it was investigated whether heme proteins could catalyze the samecarbon—silicon bond-forming reaction. Screening a panel of cytochromeP450 and myoglobin variants, product formation was observed with moreturnovers compared to the hemin and hemin with bovine serum albumin(BSA) controls, but with negligible enantioinduction (Table 5).Surprisingly, cytochrome c from Rhodothermus marinus (Rma cyt c), agram-negative, thermohalophilic bacterium from submarine hot springs inIceland (25), catalyzed the reaction with 97% ee, indicating thereaction took place in an environment where the protein exertedexcellent stereocontrol. Bacterial cytochromes c are well-studied,functionally conserved electron-transfer proteins that are not known tohave any catalytic function in living systems (26). Other bacterial andeukaryotic cytochrome c proteins also catalyzed the reaction, but withlower selectivities. Rma cyt c was selected as the platform for evolvinga carbon—silicon bond-forming enzyme.

TABLE 5 Preliminary experiments with heme and purified heme proteins

Catalyst TTN % ee Controls None 0 — Hemin  4 ± 1  0 Hemin + BSA  1 ± 1 0 P450s BM3 P450 T268A (84) 44 ± 15 0 BM3 P450 T268A C400H (121) 45 ± 3 <5 BM3 P450 CIS I263F C400S T438S 24 ± 9  <5 (122) BM3 P450 F87A T268AC400S (123) 40 ± 15 <5 BM3 P450 Hstar H92N H100N (124) 46 ± 7  0Myoglobins Sperm whale Mb 12 ± 1  0 Sperm whale Mb H64V V68A (125) 17 ±1  0 Cytochromes c Horse heart cyt c 31 ± 10 <5 Bovine heart cyt c 54 ±2  6 S. cerevisiae cyt c 11 ± 1  <5 R. marinus cyt c 34 ± 10 97 H.thermophilus cyt c  8 ± 2  16 R. globiformis cyt c  4 ± 1  <5 OthersHorse radish peroxidase 0 — C. glutamicum catalase 0 —

The crystal structure of wild-type Rma cyt c (PDB ID: 3CP5; 26) revealedthat the heme prosthetic group resides in a hydrophobic pocket, with theiron axially coordinated to a proximal His (H49) and a distal Met(M100), the latter of which is located on a loop (FIGS. 5B and 5C). Thedistal Met, common in cytochrome c proteins, is coordinately labile(27,28) and was selected for mutation based on the idea that M100 mustbe displaced upon iron-carbenoid formation, and that mutation of thisamino acid could facilitate formation of this adventitious “active site”and yield an improved carbon—silicon bond-forming biocatalyst.Therefore, a variant library made by site-saturation mutagenesis of M100was cloned and recombinantly expressed in E. coli. After proteinexpression, the bacterial cells were heat-treated (75° C. for 10minutes) before screening in the presence of phenyldimethylsilane (10mM), Me-EDA (10 mM) and sodium dithionite (Na₂S₂O₄ 10 mM) as a reducingagent, at room temperature under anaerobic conditions. The M100Dmutation stood out as highly activating: this first-generation mutantprovided chiral organosilicon (compound 3) as a single enantiomer in 550TTN, a 12-fold improvement over the wild-type protein (FIG. 5D).

Amino acid residues V75 and M103 reside close (i.e., within 7 Å) to theiron heme center of wild-type Rma cyt c. Sequential site-saturationmutagenesis at these positions in the M100D mutant led to the discoveryof triple mutant V75T M100D M103E, which catalyzed carbon—silicon bondformation in greater than 1,500 turnovers and greater than 99% ee. Thislevel of activity was more than 15 times the total turnovers reportedfor the best synthetic catalysts for this class of reaction (16). Asstand-alone mutations, both V75T and M103E were activating for wild-typeRma cyt c and the beneficial effects increased with each combination(Tables 6 and 7 and FIG. 6). Comparison of the initial reaction ratesestablished that each round of evolution enhanced the rate: relative tothe wild-type protein, the evolved triple mutant catalyzed the reactiongreater than 7-fold faster, with turnover frequency (TOF) of 46 min⁻¹(Table 7).

TABLE 6 Carbon-silicon bond formation catalyzed by Rma cyt c variants

Rma cyt c TTN WT   44 ± 27 M100D  549 ± 24 V75T  150 ± 48 M103E   70 ±21 V75T M100D  892 ± 20 V75T M100E  154 ± 37 M100D M103E  520 ± 88 V75TM100D 1518 ± 51 M103E

TABLE 7 Comparison of carbon-silicon bond forming rates of fourgenerations of Rma cyt c

Rma cyt initial rate/ turnover frequency TOF relative c variant μM min⁻¹(TOF)/min⁻¹ to WT WT  5.1 ± 0.3  6.4 ± 0.3 1 M100D 14.0 ± 0.4 17.5 ± 0.52.8 ± 0.2 V75T M100D 23.6 ± 0.8 29.5 ± 1.0 4.6 ± 0.3 V75T M100D 36.4 ±0.5 45.5 ± 0.7 7.1 ± 0.4 M103EErrors quoted in the table above are calculated from the standarddeviations of the fitting of data in the product vs time plot shown inFIG. 6.

Assaying the new enzyme against a panel of silicon and diazo reagents,it was found that the mutations were broadly activating forenantioselective carbon—silicon bond formation.

The reaction substrate scope was surveyed using heat-treated lysates ofE. coli expressing Rma cyt c V75T M100D M103E under saturatingconditions for both silane and diazo ester to determine TTN. Whereasmany natural enzymes excel at catalyzing reactions on only their nativesubstrates and little else (especially primary metabolic enzymes), thetriple mutant catalyzed the formation of twenty silicon-containingproducts, most of which were obtained cleanly as single enantiomers,showcasing the broad substrate scope of this reaction using just asingle variant of the enzyme (FIG. 7). The reaction accepted bothelectron-rich and electron-deficient silicon reagents, accommodating avariety of functional groups including ethers, aryl halides, alkylhalides, esters and amides (compounds 5-10). Silicon reagents based onnaphthalenes or heteroarenes (compounds 11-13) as well as vinyldialkyl-and trialkylsilanes could also serve as silicon donors (compounds 14,15, and 18). In addition, diazo compounds other than Me-EDA could beused for carbon—silicon bond formation (compounds 16 and 17).

The evolved Rma cyt c exhibited high specificity for carbon—silicon bondformation. Even in the presence of functional groups that could competein carbene-transfer reactions, enzymatic carbon—silicon bond formationproceeded with excellent chemoselectivity. For example, styrenylolefins, electron-rich double bonds, and terminal alkynes that are primereaction handles for synthetic derivatization were preserved under thereaction conditions, with no competing cyclopropanation orcyclopropenation activity observed. As a result, organosilicon products(compounds 12-13 and 18-20) were produced with 210 to 5,010 turnoversand excellent stereoselectivities (98 to greater than 99% ee).Preferential carbon—silicon bond formation could also be achieved withsubstrates bearing free alcohols and primary amines, affordingsilicon-containing phenol (compound 21) (910 TTN, greater than 99% ee)and aniline (compound 22) (8,210 TTN, greater than 99% ee). Thiscapability removed the need for functional group protection and/ormanipulation, offering a streamlined alternative to transition metalcatalysis for incorporating silicon into small molecules. Indeed, whenthe same reactants were subjected to rhodium catalysis (1 mol %Rh₂(OAc)₄), O—H and N—H insertions were the predominant reactionpathways, and copper catalysis (10 mol % Cu(OTf)₂) gave complex mixturesof products (Table 8). Tolerance of these highly versatilefunctionalities in enzymatic carbon—silicon bond-forming reactionsprovides opportunities for their use in downstream processing throughmetabolic engineering, bioorthogonal chemistry, and other syntheticendeavors.

TABLE 8 Rh(II)- and Cu(II)-catalyzed reactions between Me-EDA and4-(dimethylsilyl)phenol (compound 1k) or 4-(dimethylsilyl)aniline(compound 23)

Compound 21 Compound 21-iso Compound 21-di (Si—H insertion) (O—Hinsertion) (double insertion) Rh₂(OAc)₄ x ✓ ✓ Cu(OTf)₂ x x x Compound 22Compound 22-iso Compound 22-di (Si—H insertion) (N—H insertion) (doubleinsertion) Rh₂(OAc)₄ x ✓ x Cu(OTf)₂ x x x Note: x = not detected; ✓ =detected. Cu(OTf)₂ gave complex mixtures of products in both reactions.

Next, it was investigated whether all Rma cyt c variants could catalyzecarbon—silicon bond formation selectively over insertion of the carbeneinto an N—H bond in the same substrate. The evolutionary lineage wasre-visited and tested for all four generations of Rma cyt c (wild-type,M100D, V75T M100D and V75T M100D M103E) with Me-EDA and4-(dimethylsilyl)aniline (Compound 23), a reagent that could serve asboth a nitrogen and a silicon donor, to probe the proteins' bond-formingpreferences. The wild-type cytochrome c in fact exhibited a slightpreference for forming amination product (compound 24) overorganosilicon product (Compound 22). Even though silane (Compound 23)was not used for screening, and the Rma cyt c therefore never underwentdirect selection for chemoselectivity, each round of evolution effecteda distinct shift from amination to carbon—silicon bond forming activity(FIG. 8A). This evolutionary path that focused solely on increasingdesired product formation culminated in a catalyst that channeled themajority of the reactants (i.e., 97%) through carbon—silicon bondformation (i.e., greater than 30-fold improvement with respect to thewild-type). Without being bound to any particular theory, improvementwas effected by optimizing the orientation and binding of the silicondonor.

Some fungi, bacteria and algae have demonstrated promiscuous capacitiesto derivatize organosilicon molecules when these substances were madeavailable to them (1). The desire to establish silicon-basedbiosynthetic pathways led to the investigation of whether the evolvedRma cyt c could produce organosilicon products in vivo. E. coli wholecells (OD₆₀₀=15) expressing Rma cyt c V75T M100D M103E inglucose-supplemented M9-N buffer were given silane (compound 23) (0.1mmol) and Me-EDA (0.12 mmol) as neat reagents. The enzyme in thiswhole-cell system catalyzed carbon—silicon bond formation with 3,410turnovers, affording organosilicon product (compound 22) in 70% isolatedyield (greater than 95% yield based on recovered silane (compound 23))and 98% ee (FIG. 8B). These in vitro and in vivo examples ofcarbon—silicon bond formation using enzymes of the present invention andearth-abundant iron show that compositions and methods of the presentinvention are useful for efficiently forging chemical bonds notpreviously found in biology, thereby granting access to areas ofchemical space which living systems have not yet explored.

Materials and Methods

Chemicals and Reagents

Unless otherwise noted, all chemicals and reagents for chemicalreactions were obtained from commercial suppliers (Acros, ArchBioscience, Fisher Scientific, Sigma-Aldrich, TCI America) and usedwithout further purification. The following proteins were all purchasedfrom Sigma-Aldrich: bovine serum albumin (BSA), cytochrome c (frombovine, equine heart and S. cerevisiae), peroxidase II (fromhorseradish), and catalase (from C. glutamicum). Silica gelchromatography purifications were carried out using AMD Silica Gel 60,230-400 mesh. ¹H and ¹³C NMR spectra were recorded on a Bruker Prodigy400 MHz instrument and were internally referenced to the residualsolvent peak (chloroform). ²⁹Si NMR spectra were recorded on the sameinstrument and referenced to tetramethoxysilane (δ−78.9 ppm). Data for¹H NMR reported in the conventional form: chemical shift (δ ppm),multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, hept=heptet,m=multiplet, br=broad, app=appears as), coupling constant (Hz),integration. Data for ¹³C and ²⁹Si reported in terms of chemical shift(δ ppm). High-resolution mass spectra were obtained with a JEOL JMS-600HHigh Resolution Mass Spectrometer at the California Institute ofTechnology Mass Spectral Facility. Sonication was performed using aQsonica Q500 sonicator. Chemical reactions were monitored using thinlayer chromatography (Merck 60 silica gel plates) and a UV-lamp forvisualization. Gas chromatography (GC) analyses were carried out using aShimadzu GC-17A gas chromatograph, a FID detector, and J&W HP-5 (30m×0.32 mm, 0.25 μm film; 90° C. hold 1 minute, 90 to 110° C. at 15°C./minute, 110 to 280° C. at 60° C./minute, 280° C. hold 1 minute, 6.2minutes total). Analytical chiral supercritical fluid chromatography(SFC) was performed with a JACSO 2000 series instrument using i-PrOH andsupercritical CO₂ as the mobile phase, with visualization at 210 nm. Thefollowing chiral columns were used: Daicel Chiralpak IC, Chiralpak AD-H,or Chiralcel OD-H (4.6 mm×25 cm).

Plasmid pET22 was used as a cloning vector, and cloning was performedusing Gibson assembly (103). The cytochrome c maturation plasmid pEC86(104) was used as part of a two-plasmid system to express prokaryoticcytochrome c proteins. Cells were grown using Luria-Bertani medium orHyperBroth (AthenaES) with 100 μg/mL ampicillin and 20 μg/mLchloramphenicol (LB_(amp/chlor) or HB_(amp/chlor)). Cells without thepEC86 plasmid were grown with 100 μg/mL ampicillin (LB_(amp) orHB_(amp)). Electrocompetent Escherichia coli cells were preparedfollowing the protocol of Sambrook et al. (105). T5 exonuclease, Phusionpolymerase, and Taq ligase were purchased from New England Biolabs (NEB,Ipswich, Mass.). M9-N minimal medium (abbreviated as M9-N buffer; pH7.4) was used as a buffering system for whole cells, lysates, andpurified proteins, unless otherwise specified. M9-N buffer was usedwithout a carbon source; it contains 47.7 mM Na₂HPO₄, 22.0 mM KH₂PO₄,8.6 mM NaCl, 2.0 mM MgSO₄, and 0.1 mM CaCl₂.

Plasmid Construction

All variants described in this example were cloned and expressed usingthe pET22(b)+ vector (Novagen). The gene encoding Rma cyt c (UNIPROT IDB3FQS5) was obtained as a single gBlock (IDT), codon-optimized for E.coli, and cloned using Gibson assembly (103) into pET22(b)+ (Novagen)between restriction sites NdeI and XhoI in frame with an N-terminal pelBleader sequence (to ensure periplasmic localization and propermaturation; MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO:3)) and a C-terminal6xHis-tag. This plasmid was co-transformed with the cytochrome cmaturation plasmid pEC86 (104) into E. cioni® EXPRESS BL21(DE3) cells(Lucigen).

Cytochrome c Expression and Purification

Purified cytochrome c proteins were prepared as follows. One literHB_(amp/chlor) in a 4 L flask was inoculated with an overnight culture(20 mL, LB_(amp/chlor)) of recombinant E. cloni® EXPRESS BL21(DE3) cellscontaining a pET22(b)+ plasmid encoding the cytochrome c variant, andthe pEC86 plasmid. The culture was shaken at 37° C. and 200 rpm (nohumidity control) until the OD₆₀₀ was 0.7 (approximately 3 hours). Theculture was placed on ice for 30 minutes, and isopropylβ-D-1-thiogalactopyranoside (IPTG) and 5-aminolevulinic acid (ALA) wereadded to final concentrations of 20 μM and 200 μM respectively.

The incubator temperature was reduced to 20° C., and the culture wasallowed to shake for 20 hours at 200 rpm. Cells were harvested bycentrifugation (4° C., 15 minutes, 4,000×g), and the cell pellet wasstored at −20° C. until further use (at least 24 hours). The cell pelletwas resuspended in buffer containing 100 mM NaCl, 20 mM imidazole, and20 mM Tris-HCl buffer (pH 7.5 at 25° C.) and cells were lysed bysonication (2 minutes, 2 seconds on, 2 seconds off, 40% duty cycle;Qsonica Q500 sonicator). Cell lysate was placed in a 75° C. heat bathfor 10 minutes, and cell debris was removed by centrifugation for 20minutes (5000×g, 4° C.). Supernatant was sterile filtered through a 0.45μm cellulose acetate filter and purified using a 1 mL Ni-NTA column(HisTrap HP, GE Healthcare, Piscataway, N.J.) using an AKTA purifierFPLC system (GE healthcare). The cytochrome c protein was eluted fromthe column by running a gradient from 20 to 500 mM imidazole over 10column volumes.

The purity of the collected cytochrome c fractions was analyzed usingsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Pure fractions were pooled and concentrated using a 3 kDa molecularweight cut-off centrifugal filter and dialyzed overnight into 0.05 Mphosphate buffer (pH=7.5) using 3 kDa molecular weight cut-off dialysistubing. The dialyzed protein was concentrated again, flash-frozen on dryice, and stored at −20° C.

The concentration of cytochrome c was determined in triplicate using theferrous assay described below.

P450 and Globin Expression and Purification

Purified P450s and globins were prepared differently from the cytochromec proteins, described as follows. One liter HB_(amp) in a 4 L flask wasinoculated with an overnight culture (20 mL, LB_(amp)) of recombinant E.cloni® EXPRESS BL21(DE3) cells containing a pET22(b)+ plasmid encodingthe P450 or globin variant. The culture was shaken at 37° C. and 200 rpm(no humidity control) until the OD₆₀₀ was 0.7 (approximately 3 hours).The culture was placed on ice for 30 minutes, and IPTG and 5-ALA wereadded to final concentrations of 0.5 mM and 1 mM, respectively. Theincubator temperature was reduced to 20° C., and the culture was allowedto shake for 20 hours at 200 rpm. Cells were harvested by centrifugation(4° C., 15 minutes, 4,000×g), and the cell pellet was stored at −20° C.until further use (at least 24 hours). The cell pellet was resuspendedin buffer containing 100 mM NaCl, 20 mM imidazole, and 20 mM Tris-HClbuffer (pH 7.5 at 25° C.). Hemin (30 mg/mL, 0.1 M NaOH; FrontierScientific) was added to the resuspended cells such that 1 mg of heminwas added for every 1 gram of cell pellet. Cells were lysed bysonication (2 minutes, 1 seconds on, 2 seconds off, 40% duty cycle;Qsonica Q500 sonicator). Cell debris was removed by centrifugation for20 minutes (27,000×g, 4° C.). Supernatant was sterile filtered through a0.45 μm cellulose acetate filter, and purified using a 1 mL Ni-NTAcolumn (HisTrap HP, GE Healthcare, Piscataway, N.J.) using an AKTApurifier FPLC system (GE healthcare). The P450 and globin proteins wereeluted from the column by running a gradient from 20 to 500 mM imidazoleover 10 column volumes.

The purity of the collected protein fractions was analyzed usingSDS-PAGE. Pure fractions were pooled and concentrated using a 10 kDamolecular weight cut-off centrifugal filter and buffer-exchanged with0.1 M phosphate buffer (pH=8.0). The purified protein was flash-frozenon dry ice and stored at −20° C.

P450 and globin concentrations were determined in triplicate using thehemochrome assay described below.

Hemochrome Assay

A solution of sodium dithionite (10 mg/mL) was prepared in M9-N buffer.Separately, a solution of 1 M NaOH (0.4 mL) was mixed with pyridine (1mL), followed by centrifugation (10,000×g, 30 seconds) to separate theexcess aqueous layer gave a pyridine-NaOH solution. To a cuvettecontaining 700 μL protein solution (purified protein or heat-treatedlysate) in M9-N buffer, 50 μL of dithionite solution and 250 μLpyridine-NaOH solution were added. The cuvette was sealed with Parafilm,and the UV-Vis spectrum was recorded immediately. Cytochrome cconcentration was determined using ϵ₅₅₀₋₅₃₅=22.1 mM⁻¹cm⁻¹ (106). Proteinconcentrations determined by the hemochrome assay were in agreement withthat determined by the bicinchoninic acid (BCA) assay (Thermo Fisher)using bovine serum albumin (BSA) for standard curve preparation.

Ferrous Assay

To a cuvette containing 700 μL protein solution in M9-N buffer was added50 μL of dithionite solution (10 mg/mL in M9-N buffer). The cuvette wassealed with Parafilm, and the UV-Vis spectrum was recorded immediately.The absorbance value for the peak at 550 nm was recorded, and backgroundabsorbance at 600 nm was subtracted. Using the protein concentration asdetermined by the hemochrome assay, ferrous ϵ₅₅₀₋₆₀₀ was determined tobe 27 mM⁻¹cm⁻¹ for wild-type Rma cyt c, and 21 mM⁻¹cm⁻¹ for Rma V75TM100D M103E (see, FIGS. 9A and 9B). Concentrations of Rma M100D and V75TM100D were determined using the extinction coefficient calculated forV75T M100D M103E.

Library Construction

Cytochrome c site-saturation mutagenesis libraries were generated usinga modified version of the 22-codon site-saturation method (107). Foreach site-saturation library, oligonucleotides were ordered such thatthe coding strand contained the degenerate codon NDT, VHG or TGG. Thereverse complements of these primers were also ordered. The threeforward primers were mixed together in a 12:9:1 ratio, (NDT:VHG:TGG) andthe three reverse primers were mixed similarly. Two PCR reactions wereperformed, pairing the mixture of forward primers with a pET22(b)+internal reverse primer, and the mixture of reverse primers with apET22b internal forward primer. The two PCR products were gel purified,ligated together using Gibson assembly (103), and transformed into E.cloni® EXPRESS BL21(DE3) cells.

Enzyme Library Screening

Single colonies were picked with toothpicks off of LB_(amp/chlor) agarplates, and grown in deep-well (2 mL) 96-well plates containingLB_(amp/chlor) (400 μL) at 37° C., 250 rpm shaking, and 80% relativehumidity overnight. After 16 hours, 30 μL aliquots of these overnightcultures were transferred to deep-well 96-well plates containingHB_(amp/chlor) (1 mL) using a 12-channel EDP3-Plus 5-50 μL pipette(Rainin). Glycerol stocks of the libraries were prepared by mixing cellsin LB_(amp/chlor) (100 μL) with 50% v/v glycerol (100 μL). Glycerolstocks were stored at −78° C. in 96-well microplates. Growth plates wereallowed to shake for 3 hours at 37° C., 250 rpm shaking, and 80%relative humidity. The plates were then placed on ice for 30 minutes.Cultures were induced by adding 10 μL of a solution, prepared in steriledeionized water, containing 2 mM isopropyl β-D-1-thiogalactopyranoside(IPTG) and 20 mM ALA. The incubator temperature was reduced to 20° C.,and the induced cultures were allowed to shake for 20 hours (250 rpm, nohumidity control). Cells were pelleted (4,000×g, 5 min, 4° C.) andresuspended in 500 μL M9-N buffer. For cell lysis, plates were placed ina 75° C. water bath for 10 minutes, followed by centrifugation (4,000×g,5 min, 4° C.) to remove cell debris. The resulting heat-treated lysates(340 μL) were then transferred to deep-well plates for biocatalyticreactions. In an anaerobic chamber, to deep-well plates of heat-treatedlysates were added Na₂S₂O₄ (40 μL per well, 100 mM in dH₂O), PhMe₂SiH(10 μL per well, 400 mM in MeCN) and Me-EDA (10 μL per well, 400 mM inMeCN). The plates were sealed with aluminum sealing tape, removed fromthe anaerobic chamber, and shaken at 400 rpm for 1.5 hours. Afterquenching with cyclohexane (1 mL), internal standard was added (20 μL of20 mM methyl 2-phenylacetate in cyclohexane) and the reaction mixtureswere pipetted up and down to thoroughly mix the organic and aqueouslayers. The plates were centrifuged (4,000×g, 5 minutes) and the organiclayer (400 μL) was transferred to shallow-well 96-well plates for SFCanalysis. Hits from library screening were confirmed by small-scalebiocatalytic reactions, which were analyzed by GC and SFC for accuratedetermination of turnovers and enantioselectivities.

Protein Lysate Preparation

Protein lysates for biocatalytic reactions were prepared as follows: E.coli cells expressing Rma cyt c variants were pelleted (4,000×g, 5minutes, 4° C.), resuspended in M9-N buffer and adjusted to theappropriate OD₆₀₀. The whole-cell solution was heat-treated (75° C. for10 minutes) then centrifuged (14,000×g, 10 minutes, 4° C.) to removecell debris. The supernatant was sterile filtered through a 0.45 μmcellulose acetate filter into a 6 mL crimp vial, crimp sealed, and thehead space of the crimp vial was degassed by bubbling argon through forat least 10 minutes. The concentration of cytochrome c protein lysatewas determined using the ferrous assay described above. Using thisprotocol, the protein concentrations typically observed for OD₆₀₀=15lysates were in the 8-15 μM range for wild-type Rma cyt c and 2-10 μMfor other Rma cyt c variants.

Preliminary Experiments with Heme and Purified Heme Proteins

Commercially available heme proteins were screened to identify the mostenantioselective protein variant as a starting point for directedevolution. Experiments with heme proteins were performed using 10 μMpurified heme protein, 10 mM silane, 10 mM diazo ester, 10 mM Na₂S₂O₄, 5vol % MeCN, M9-N buffer at room temperature under anaerobic conditionsfor 1.5 hours. Experiments with hemin were performed using 100 μM hemin.Experiments with hemin and BSA were performed using 100 μM hemin in thepresence of BSA (0.75 mg/mL). Reactions were performed in triplicate.TTNs reported as the average of three experiments. Within instrumentdetection limit, variability in % ee was not observed. Unreactedstarting materials were observed at the end of all reactions and noattempt was made to optimize these reactions.

Carbon—Silicon Bond Formation Catalyzed by Rma cyt c Variants

Experiments were performed using lysates of E. coli expressing a Rma cytc variant (OD₆₀₀=15; heat-treated at 75° C. for 10 minutes), 10 mMsilane, 10 mM diazo ester, 10 mM Na₂S₂O₄, 5 vol % MeCN, M9-N buffer atroom temperature under anaerobic conditions for 1.5 hours. Reactionswere performed in triplicate. TTNs reported as the average of threeexperiments.

Small-Scale Biocatalytic Reaction

In an anaerobic chamber, protein lysate (340 μL) in a 2 mL crimp vialwas added to 40 μL Na₂S₂O₄ (100 mM in dH₂O), 10 μL PhMe₂SiH (400 or 800mM in MeCN), and 10 μL Me-EDA (400 mM in MeCN). The vial was crimpsealed, removed from the anaerobic chamber, and shaken at 400 rpm atroom temperature for the stated reaction time. At the end of thereaction, the crimp vial was opened and the reaction was quenched withcyclohexane (1 mL). Internal standard was added (20 μL of 20 mM2-phenylethanol in cyclohexane) and the reaction mixture was transferredto a microcentrifuge tube, vortexed (10 seconds, 3 times), thencentrifuged (14,000×g, 5 min) to completely separate the organic andaqueous layers (the vortex-centrifugation step was repeated if completephase separation was not achieved). The organic layer (750 μL) wasremoved for GC and SFC analysis. All biocatalytic reactions wereperformed in triplicate unless otherwise stated. The total turnovernumbers (TTNs) reported were calculated with respect to the proteincatalyst and represented the total number of turnovers that is possibleto obtain from the catalyst under the stated reaction conditions.

Determining Carbon—Silicon Bond Formation Rates

To determine the initial reaction rate, experiments were performed usingpurified Rma cyt c variants (0.8 μM), 10 mM silane, 10 mM diazo ester,10 mM Na₂S₂O₄, 5 vol % MeCN, M9-N buffer at room temperature underanaerobic conditions for various time intervals. The data used fordetermining the initial rate of each Rma cyt c variant is shown in thegraph in FIG. 6.

For the timed experiments, the following procedure was used: in ananaerobic chamber, 1 mL Na₂S₂O₄ (100 mM in dH₂O) was added to 4 mLpurified Rma cyt c protein (2.0 μM in M9-buffer) to give Solution 1. Tofour 2 mL microcentrifuge tubes were each added 180 μL M9-N buffer, 10μL PhMe2SiH (400 mM in MeCN) and 10 μL Me-EDA (400 mM in MeCN), and themixtures were mixed thoroughly on a shaker (480 rpm for 2 min). To thesemixtures were added 200 μL Solution 1, and the microcentrifuge tubeswere closed and quickly shaken by hand for 3 seconds to ensure thoroughmixing, before the tubes were returned to the shaker. The reactions werestopped at specific time points (2 minutes, 4 minutes, 6 minutes, and 8minutes) by quick addition (within 10 seconds) of 40 μL pyridinesolution (400 mM in dH₂O), 20 μL internal standard (20 mM acetophenonein toluene) and 1 mL cyclohexane. (Note: pyridine was added as aquencher to significantly slow down the reaction.). After the mixtureswere vortexed for 20 seconds, 200 μL organic layer was immediatelyremoved for GC analysis.

Rh(II)- and Cu(II)-Catalyzed Reactions

Rh₂(OAc)₄ and Cu(OTf)₂, which are known to catalyze carbene insertioninto Si—H bonds under ligand-free conditions (126), were tested fortheir chemoselectivities towards Si—H, O—H and N—H insertions (Table 4).To a 5 mL vial was added silane (0.1 mmol, 1.0 equiv.), metal catalyst(Rh₂(OAc)₄ (0.44 mg, 1 mol %) or Cu(OTf)₂ (3.62 mg, 10 mol %)) and DCM(0.5 mL). The mixture was cooled to −78° C. before a solution of Me-EDA(25 μL, 2.0 equiv.) in DCM (0.3 mL) was added dropwise. After slowlywarming up to room temperature in 4 hours, the reaction mixture wasfiltrated through a short pad of silica, diluted with DCM, and analyzedby GC-MS.

Products isolated from Rh₂(OAc)₄-catalyzed reactions with compounds 1kand 23 are shown below. Both reactions generated multiple products,rendering product purification and quantitative analysis of thesereactions difficult. Multiple rounds of purification by silica columnchromatography were required to obtain samples suitable forcharacterization; yields were therefore not determined for thesereactions. Notably, Si—H insertion products (compounds 21 and 22) werenot observed in these reactions.

A Rh₂(OAc)₄-catalyzed reaction with compound 1k afforded Compoundrac-21-di and Compound rac-21-iso:

Compound rac-21-di

¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J=8.6 Hz, 2H), 6.87 (d, J=8.7 Hz,2H), 4.75 (q, J=6.8 Hz, 1H), 4.22 (q, J=7.1 Hz, 2H), 4.01 (q, J=7.1 Hz,2H), 2.21 (q, J=7.1 Hz, 1H), 1.62 (d, J=6.8 Hz, 3H), 1.25 (t, J=7.1 Hz,3H), 1.17-1.10 (m, 6H), 0.33 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 176.19,172.25, 158.86, 135.54, 128.29, 128.28, 114.65, 72.42, 61.46, 59.93,30.30, 18.68, 14.47, 14.28, 11.43, −3.77, −4.60. HRMS (FAB) m/z:352.1714 (M⁺); calc. for C₁₈H₂₈SiO₅: 352.1706.

Compound rac-21-iso (This Compound Was Inseparable From Impurities)

¹H NMR (400 MHz, CDCl₃) δ 7.44 (d, J=8.7 Hz, 2H), 6.87 (d, J=8.7 Hz,2H), 4.76 (q, J=6.8 Hz, 1H), 4.39 (hept, J=3.7 Hz, 1H), 4.22 (q, J=6.9Hz, 2H), 1.62 (d, J=6.8 Hz, 3H), 1.26 (t, J=7.1 Hz, 3H), 0.31 (d, J=3.8Hz, 6H).

A Rh₂(OAc)₄-catalyzed reaction with compound 23 afforded Compoundrac-22-iso and the corresponding double N—H insertion product:

Compound rac-22-iso

¹H NMR (400 MHz, CDCl₃) δ 7.24-7.17 (m, 2H), 6.88-6.68 (m, 3H), 4.68(dq, J=5.5, 2.8 Hz, 1H), 4.22-4.11 (m, 3H), 1.50 (br d, J=6.4 Hz, 3H),1.25 (t, J=7.1 Hz, 3H), 0.19 (d, J=2.8 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃)δ 173.94, 135.20, 129.50, 119.78, 114.93, 61.47, 53.20, 18.62, 14.31,0.71. FIRMS (FAB) m/z: 250.1253 ((M+H)—H₂ ⁺); calc. for C₁₃H₂₀SiNO₂:250.1263.

Interestingly, the following compound was also isolated as a mixture ofdiastereomers. The counter anion of the ammonium salt was notdetermined.

¹H NMR (400 MHz, CDCl₃) δ 7.22 (overlapping doublets, J=8.8, 7.3 Hz,2H), 6.84 (t, J=7.3 Hz, 1H), 6.78 (br d, J=7.9 Hz, 2H), 4.99-4.55 (m,1H), 4.44 (q, J=7.1 Hz, 2H), 4.23 (app qd, J=7.2, 2.0 Hz, 4H), 1.55 (d,J=7.2 Hz, 6H), 1.29 (t, J=7.1 Hz, 6H), 0.30-0.19 (m, 6H). ¹³C NMR (100MHz, CDCl₃) δ 174.40, 146.38, 128.89, 119.30, 117.17, 61.24, 56.24,15.95, 14.34, 0.71. FIRMS (FAB) m/z: 352.1958 (M^(t)); calc. forC₁₈H₃₀SiNO₄: 352.1944.

Substrate Synthesis and Characterization

The following commercially available substrates were used as received:phenyldimethylsilane (Sigma-Aldrich), benzyldimethylsilane(Sigma-Aldrich), and ethyl 2-diazopropanoate (Arch Bioscience). Thefollowing diazo compounds were prepared according to literatureprocedures: isopropyl 2-diazopropanoate (128), ethyl 2-diazobutanoate(129). The preparation and characterization of Compounds 1b, 1c, 1d, 1e,1f, 1g, 1h, 1i, 1k, 1ks, 23, 23s, 1m, 1n, 1o, 1p, 1q, 1r are describedbelow.

Dimethyl(p-tolyl)silane (Compound 1b)

In a 100 mL round-bottom flask, chlorodimethylsilane (1.11 mL, 10.0mmol) in THF (6 mL) was cooled to 0° C. A solution of4-methylphenylmagnesium bromide (24 mL, 0.5 M in THF) was added dropwiseslowly over 15 minutes. Then the reaction was allowed to warm to roomtemperature and stirred for 8 hours. The reaction mixture was quenchedwith NH₄Cl (5 mL, sat. aq.) and the product was extracted with Et₂O (15mL×3). The organic layer was washed with water (20 mL), then brine (20mL), then dried over MgSO₄ and concentrated under reduced pressure (200torr). The crude product was purified by silica column chromatographywith pentane to afford Compound 1b (1.27 g, 8.44 mmol, 84%). Thiscompound is known (130).

¹H NMR (400 MHz, CDCl₃) δ 7.48 (d, J=7.8 Hz, 2H), 7.23 (d, J=7.4 Hz,2H), 4.46 (hept, J=3.7 Hz, 1H), 2.39 (s, 3H), 0.37 (d, J=3.7 Hz, 6H).

(4-Methoxyphenyl)dimethylsilane (Compound 1c)

In a 100 mL round-bottom flask, chlorodimethylsilane (1.11 mL, 10.0mmol) in THF (6 mL) was cooled to 0° C. A solution of4-methoxyphenylmagnesium bromide (24 mL, 0.5 M in THF) was addeddropwise slowly over 15 minutes. Then the reaction was allowed to warmto room temperature and stirred for 8 hours. The reaction mixture wasquenched with NH₄Cl (5 mL, sat. aq.) and the product was extracted withEt₂O (15 mL×3). The organic layer was washed with water (20 mL), thenbrine (20 mL), then dried over MgSO₄ and concentrated under reducedpressure (200 torr). The crude product was purified by silica columnchromatography with pentane/Et₂O (10:1) to afford Compound 1c (1.60 g,9.62 mmol, 96%). This compound is known (130). ¹H NMR (400 MHz, CDCl₃) δ7.51 (d, J=8.7 Hz, 2H), 6.96 (d, J=8.6 Hz, 2H), 4.75-4.19 (m, 1H), 3.85(s, 3H), 0.37 (d, J=3.8 Hz, 6H).

(4-Chlorophenyl)dimethylsilane (Compound 1d)

In a 100 mL round-bottom flask, chlorodimethylsilane (1.11 mL, 10.0mmol) in THF (6 mL) was cooled to 0° C. A solution of4-chlorophenylmagnesium bromide (24 mL, 0.5 M in THF) was added dropwiseslowly over 15 minutes. Then the reaction was allowed to warm to roomtemperature and stirred for 8 hours. The reaction mixture was quenchedwith NH₄Cl (5 mL, sat. aq.) and the product was extracted with Et₂O (15mL×3). The organic layer was washed with water (20 mL), then brine (20mL), then dried over MgSO₄ and concentrated under reduced pressure (200torr). The crude product was purified by silica column chromatographywith pentane to afford Compound 1d (1.27 g, 7.45 mmol, 75%). Thiscompound is known (130).

¹H NMR (400 MHz, CDCl₃) δ 7.46 (d, J=8.3 Hz, 2H), 7.34 (d, J=8.2 Hz,2H), 4.41 (hept, J=3.8 Hz, 1H), 0.34 (d, J=3.7 Hz, 6H).

(4-(Trifluoromethyl)phenyl)dimethylsilane (Compound 1e)

In a 100 mL round-bottom flask, 1-bromo-4-(trifluoromethyl)benzene (1.4mL, 10.0 mmol) in THF (15 mL) was cooled to −78° C. n-BuLi (7.5 mL, 1.6M in hexane) was added dropwise slowly over 15 minutes. The resultingmixture was stirred at −78° C. for 2 hours before the dropwise additionof chlorodimethylsilane (1.0 mL, 9.0 mmol). The reaction was allowed towarm to room temperature and stirred for 8 hours. The reaction mixturewas quenched with NH₄Cl (5 mL, sat. aq.) and the product was extractedwith Et₂O (15 mL×3). The organic layer was washed with water (20 mL),then brine (20 mL), then dried over MgSO₄ and concentrated under reducedpressure (200 torr). The crude product was purified by silica columnchromatography with pentane to afford Compound 1e (0.81 g, 3.97 mmol,40%). This compound is known (130). ¹H NMR (400 MHz, CDCl₃) δ 7.66 (d,J=7.7 Hz, 2H), 7.60 (d, J=7.9 Hz, 2H), 4.46 (hept, J=3.8 Hz, 1H), 0.38(d, J=3.8 Hz, 6H).

(4-(Chloromethyl)phenyl)dimethylsilane (Compound 1f)

In a 250 mL round-bottom flask, (4-bromophenyl)methanol (5.61 g, 30.0mmol) in THF (100 mL) was cooled to −78° C. n-BuLi (30.0 mL, 2.5 M inhexane) was added dropwise slowly over 30 minutes. The resulting mixturewas stirred at −78° C. for 2 hours before the dropwise addition ofchlorodimethylsilane (4.5 mL, 40.0 mmol). The reaction was allowed towarm to room temperature and stirred for 8 hours. The reaction mixturewas quenched with NH₄Cl (50 mL, sat. aq.) and the product was extractedwith DCM (50 mL×3). The organic layer was washed with water (20 mL),then brine (20 mL), then dried over MgSO₄ and concentrated under reducedpressure (100 torr). The crude product was purified by silica columnchromatography with EtOAc/hexane (1:3) to afford(4-(dimethylsilyl)phenyl)methanol (2.96 g, 17.8 mmol, 59%). Thiscompound is known (131). ¹H NMR (400 MHz, CDCl₃) δ 7.55 (d, J=8.0 Hz,2H), 7.37 (d, J=8.1 Hz, 2H), 4.70 (s, 2H), 4.43 (hept, J=3.7 Hz, 1H),0.35 (d, J=3.8 Hz, 6H).

To a solution of (4-(dimethylsilyl)phenyl)methanol (498.9 mg, 3.0 mmol)in DCM (4 mL) were added triethylamine (0.5 mL, 3.6 mmol) and4-methylbenzenesulfonyl chloride (629.1 mg, 3.3 mmol). The reactionmixture was stirred at room temperature for 8 hours. The reaction wasthen diluted with DCM (10 mL) and washed with water (20 mL), then brine(20 mL), then dried over MgSO₄ and the organic layer was concentratedunder reduced pressure (100 torr). The crude product was purified bysilica column chromatography with pentane to afford Compound if (0.22 g,1.19 mmol, 40%). ¹H NMR (400 MHz, CDCl₃) δ 7.55 (d, J=8.0 Hz, 2H), 7.39(d, J=7.9 Hz, 2H), 4.59 (s, 2H), 4.43 (hept, J=3.8 Hz, 1H), 0.35 (d,J=3.8 Hz, 6H). FIRMS (FAB) m/z: 183.0399 ((M+H)—H₂ ⁺); calc. forC₉H₁₂SiCl: 183.0397.

Methyl 4-(dimethylsilyl)benzoate (Compound 1g)

In a 100 mL round-bottom flask, methyl 4-iodobenzoate (2.62 g, 10.0mmol) in THF (15 mL) was cooled to −78° C. i-PrMgC1 (6 mL, 2.0 M inEt₂O) was added dropwise slowly over 5 minutes. The resulting mixturewas allowed to warm to −40° C. in 2 hours and maintained at −40° C. foranother 2 hours before the dropwise addition of chlorodimethylsilane(1.2 mL, 11.0 mmol). The reaction was allowed to warm to roomtemperature and stirred for 8 hours. The reaction mixture was quenchedwith NH₄Cl (5 mL, sat. aq.) and the product was extracted with DCM (15mL×3). The organic layer was washed with water (20 mL), then brine (20mL), then dried over MgSO₄ and concentrated under reduced pressure (100torr). The mixture was dissolved in Et₂O (5 mL) and treated with hexane(25 mL). This crashed out most of the starting material, which wasremoved by filtration. The filtrate was collected, concentrated underreduced pressure, and purified by silica column chromatography withEtOAc/hexane (1:20) to afford Compound 1g (0.88 g, 4.53 mmol, 45%). ¹HNMR (400 MHz, CDCl₃) δ 8.00 (d, J=8.1 Hz, 2H), 7.62 (d, J=8.2 Hz, 2H),4.45 (hept, J=3.8 Hz, 1H), 3.92 (s, 3H), 0.37 (d, J=3.8 Hz, 6H). ¹³C NMR(100 MHz, CDCl₃) δ 167.35, 143.92, 134.13, 130.78, 128.72, 52.27, −3.84.FIRMS (FAB) m/z: 195.0843 (M+H⁺); calc. for C₁₀H₁₅SiO₂: 195.0841.

4-(dimethylsilyl)-N,N-dimethylbenzamide (Compound 1h)

To a solution of dimethylamine hydrochloride (2.45 g, 30.0 mmol) in DCM(50 mL) was added triethylamine (4.2 mL, 30.0 mmol). The mixture wasstirred for 30 minutes before the addition of 4-iodobenzoic acid (6.20g, 25.0 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC,4.66 g, 30.0 mmol). The reaction was stirred for 8 hours at roomtemperature. Then the reaction mixture was washed with water (50 mL),HCl (aq., 1 M, 50 mL), NaHCO₃ (sat. aq., 50 mL), and brine (50 mL), thendried over MgSO₄ and concentrated under reduced pressure (50 torr). Thecrude product was dissolved in Et₂O (5 mL) and treated with hexane (50mL). The target product 4-iodo-N,N-dimethylbenzamide (6.88 g, 25.0 mmol,quantitative) crashed out and was collected by filtration.

In a 100 mL round-bottom flask, 4-iodo-N,N-dimethylbenzamide (1.65 g,6.0 mmol) in

THF (15 mL) was cooled to −78° C. i-PrMgC1 (6 mL, 2.0 M in Et₂O) wasadded dropwise slowly over 5 minutes. The resulting mixture was allowedto warm to −40° C. within 2 hours and maintained at −40° C. for another2 hours before the dropwise addition of chlorodimethylsilane (1.3 mL,12.0 mmol). The reaction was allowed to warm to room temperature andstirred for 8 hours. The reaction mixture was quenched with NH₄Cl (5 mL,sat. aq.) and the product was extracted with DCM (15 mL×3). The organiclayer was washed with water (20 mL), then brine (20 mL), then dried overMgSO₄ and concentrated under reduced pressure (50 torr). The mixture wasdissolved in Et₂O (5 mL) and treated with hexane (25 mL). Most of thestarting material crashed out and was removed by filtration. Thefiltrate was collected, concentrated under reduced pressure, and thenpurified by silica column chromatography with EtOAc/hexane (1:2) toafford Compound 1h (0.12 g, 0.579 mmol, 10%). ¹H NMR (400 MHz, CDCl₃) δ7.57 (d, J=8.3 Hz, 2H), 7.39 (d, J=8.3 Hz, 2H), 4.43 (hept, J=3.8 Hz,1H), 3.05 (s, 6H), 0.35 (d, J=3.7 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ171.74, 139.53, 137.04, 134.11, 126.42, −3.74. HRMS (FAB) m/z: 208.1153(M+H⁺); calc. for C₁₁H₁₈ONSi: 208.1158.

(3,4-Dihydro-2H-pyran-6-yl)dimethylsilane (Compound 1i)

In a 100 mL round-bottom flask, 3,4-dihydro-2H-pyran (2.00 g, 24.0 mmol)in THF (1.0 mL) and pentane (40 mL) was cooled to −78° C. t-BuLi (15.5mL, 1.7 M in pentane) was added dropwise slowly over 20 minutes. Theresulting mixture was allowed to warm to 0° C. within 2 hours andmaintained at 0° C. for another 2 hours before the dropwise addition ofchlorodimethylsilane (2.6 mL, 24.0 mmol). The reaction was allowed towarm to room temperature and stirred for 8 hours. The reaction mixturewas quenched with NH₄Cl (5 mL, sat. aq.) and the product was extractedwith Et₂O (15 mL×3). The organic layer was washed with water (20 mL),then brine (20 mL), then dried over MgSO₄ and concentrated under reducedpressure (200 torr). The crude product was purified by silica columnchromatography with Et₂O/pentane (1:30) to afford Compound 1i (2.80 g,19.7 mmol, 82%). This compound is known (132). ¹H NMR (400 MHz, CDCl₃) δ5.09 (t, J=3.8 Hz, 1H), 4.05-3.91 (m, 3H), 2.04 (td, J=6.4, 3.8 Hz, 2H),1.92-1.83 (m, 2H), 0.19 (d, J=3.8 Hz, 6H).

4-(Dimethylsilyl)phenol (Compound 1k)

To a solution of 4-bromophenol (1.73 g, 10.0 mmol) in THF (15 mL) wasadded NaH (60% in mineral oil, 0.48 g, 12.0 mmol). After the mixture wasstirred for 30 minutes, it was cooled down to −78° C. t-BuLi (6 mL, 1.7M in pentane) was added dropwise slowly over 15 minutes. The resultingmixture was stirred at −78° C. for 2 hours before the dropwise additionof chlorodimethylsilane (2.3 mL, 21.0 mmol). The reaction was allowed towarm to room temperature and stirred for 8 hours. The reaction mixturewas quenched with NH₄Cl (5 mL, sat. aq.) and the product was extractedwith DCM (15 mL×3). The organic layer was washed with water (20 mL),then brine (20 mL), then dried over MgSO₄ and concentrated under reducedpressure (50 torr). The crude product was purified by silica columnchromatography with EtOAc/hexane (1:7) to afford Compound 1k (0.60 g,3.94 mmol, 39%). This compound is known (133). ¹H NMR (400 MHz, CDCl₃) δ7.43 (d, J=8.5 Hz, 2H), 6.85 (d, J=8.5 Hz, 2H), 4.78 (s, 1H), 4.40(hept, J=3.7 Hz, 1H), 0.32 (d, J=3.8 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ156.61, 135.82, 128.66, 115.16, −3.38.

(4-(Benzyloxy)phenyl)dimethylsilane (Compound 1ks)

To a solution of 4-bromophenol (5.19 g, 30.0 mmol) in MeCN (50 mL) wasadded K₂CO₃ (5.53 g, 40.0 mmol). After the mixture was stirred at 40° C.for 30 minutes, BnBr (3.6 mL, 30.0 mmol) was added over 2 minutes. Theresulting mixture was stirred at 40° C. for 3 hours. The reactionmixture was washed with water (20 mL), then brine (20 mL), then driedover MgSO₄ and concentrated under reduced pressure (50 torr) to affordthe crude product 1-(benzyloxy)-4-bromobenzene (7.85 g, 30.0 mmol,quantitative).

In a 100 mL round-bottom flask, 1-(benzyloxy)-4-bromobenzene (6.57 g,25.0 mmol) in THF (15 mL) was cooled to −78° C. t-BuLi (19.0 mL, 1.7 Min pentane) was added dropwise slowly over 15 minutes. The resultingmixture was stirred at −78° C. for 2 hours before the dropwise additionof chlorodimethylsilane (3.6 mL, 32.5 mmol). The reaction was allowed towarm to room temperature and stirred for 8 hours. The reaction mixturewas quenched with NH₄Cl (5 mL, sat. aq.) and the product was extractedwith Et₂O (15 mL×3). The organic layer was washed with water (20 mL),then brine (20 mL), then dried over MgSO₄ and concentrated under reducedpressure (50 torr). The crude product was purified by silica columnchromatography with pentane to afford Compound 1ks (4.02 g, 16.6 mmol,66%). ¹H NMR (400 MHz, CDCl₃) δ 7.51-7.27 (m, 7H), 7.00 (d, J=8.6 Hz,2H), 5.08 (s, 2H), 4.41 (hept, J=3.7 Hz, 1H), 0.32 (d, J=3.7 Hz, 6H).FIRMS (FAB) m/z: 241.1053 ((M+H)—H₂ ⁺); calc. for C₁₄H₁₇OSi: 241.1049.

4-(Dimethylsilyl)aniline (Compound 23)

To a solution of 4-bromoaniline (3.44 g, 20.0 mmol) in DCM (30 mL) wasadded triethylamine (5.6 mL, 20.0 mmol) and N,N-dimethylpyridin-4-amine(DMAP, 244.3 mg, 2.0 mmol). After the mixture was stirred for 30minutes, 1,2-bis(chlorodimethylsilyl)ethane (4.78 g, 20.0 mmol) wasadded in one portion. The reaction was stirred at 40° C. for 3 hours.The reaction mixture was filtrated through a pad of dry Celite quicklyto remove the triethylamine hydrochloride. The resulting solution wasconcentrated under reduced pressure (50 torr) to afford the crudeproduct 1-(4-bromophenyl)-2,2,5,5-tetramethyl-1,2,5-azadisilolidine.Note that this compound is moisture sensitive and decomposes on silica.

The crude 1-(4-bromophenyl)-2,2,5,5-tetramethyl-1,2,5-azadisilolidine inTHF (30 mL) in a 100 mL round-bottom flask was cooled to −78° C. t-BuLi(17.6 mL, 1.7 M in pentane) was added dropwise slowly over 15 minutes.The resulting mixture was stirred at −78° C. for 2 hours before thedropwise addition of chlorodimethylsilane (3.3 mL, 30.0 mmol). Thereaction was allowed to warm to room temperature and stirred for 8hours. The reaction mixture was treated with Et₂O (50 mL) to allow theinorganic salts to crash out. The suspension was filtrated through a padof Celite and basic alumina (1:1 mixture). The resulting solution wasconcentrated under reduced pressure (50 torr). The crude product wasloaded on silica and allowed to sit for 15 minutes (for removal of thenitrogen protecting group), before purification by silica columnchromatography with EtOAc/hexane (1:7) to afford Compound 23 (1.21 g,8.00 mmol, 40%). This compound is known (134). ¹H NMR (400 MHz, CDCl₃) δ7.38 (d, J=8.4 Hz, 2H), 6.83 (d, J=8.4 Hz, 2H), 4.84 (s, 2H), 4.38(hept, J=3.9 Hz, 1H), 0.30 (d, J=3.7 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ144.82, 135.48, 127.64, 116.07, −3.38.

Benzyl (4-(dimethylsilyl)phenyl)carbamate (Compound 23s)

To a solution of 4-(dimethylsilyl)aniline (151.2 mg, 1.0 mmol) in DCM (3mL) was added pyridine (162 μL, 2.0 mmol). After the mixture was stirredfor 10 minutes, benzyl carbonochloridate (CbzCl, 170 μL, 1.2 mmol) wasadded in one portion. After stirring at room temperature overnight, thereaction mixture was washed with water (20 mL), then brine (20 mL), thendried over MgSO₄ and concentrated under reduced pressure (50 torr). Thecrude product was purified by silica column chromatography withEtOAc/hexane (1:7) to afford Compound 23s (130.4 mg, 0.457 mmol, 46%).¹H NMR (400 MHz, CDCl₃) δ 7.50 (d, J=8.4 Hz, 2H), 7.46-7.33 (m, 5H),6.68 (s, 1H), 5.23 (s, 2H), 4.42 (hept, J=3.7 Hz, 1H), 0.34 (d, J=3.7Hz, 6H). HRMS (FAB) m/z: 286.1274 (M+H⁺); calc. for C₁₆H₂₀O₂NSi:286.1263.

(4-Ethynylphenyl)dimethylsilane (Compound 1m)

A solution of ((4-bromophenyl)ethynyl)trimethylsilane (5.06 g, 20.0mmol) and K₂CO₃ (5.53 g, 40.0 mmol) in MeOH (40 mL) was stirred at roomtemperature for 4 hours. MeOH was removed under reduced pressure, andthe crude product was washed with water (30 mL) and extracted with Et₂O(40 mL). The organic layer was dried over MgSO₄, filtrated through a padof silica, and concentrated under reduced pressure to afford the product1-bromo-4-ethynylbenzene (3.21 g, 17.7 mmol, 89%).

In a 100 mL round-bottom flask, 1-bromo-4-ethynylbenzene (1.81 g, 10.0mmol) in THF (15 mL) was cooled to −78° C. n-BuLi (8.0 mL, 2.5 M inhexane) was added dropwise very slowly over 30 minutes. The resultingmixture was stirred at −78° C. for 2 hours before the dropwise additionof a solution of chlorodimethylsilane (1.1 mL, 10.0 mmol) in THF (20mL). The reaction was allowed to warm to room temperature and stirredfor 8 hours. The reaction mixture was quenched with NH₄Cl (5 mL, sat.aq.) and the product was extracted with Et₂O (15 mL×3). The organiclayer was washed with water (20 mL), then brine (20 mL), then dried overMgSO₄ and concentrated under reduced pressure (200 torr). The crudeproduct was purified by distillation under reduced pressure (1.4 torr)at 45° C. (1.12 g, 6.99 mmol, 70%). This compound is known (135). ¹H NMR(400 MHz, CDCl₃) δ 7.49 (d, J=3.9 Hz, 4H), 4.42 (hept, J=3.8 Hz, 1H),3.10 (s, 1H), 0.34 (d, J=3.8 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 138.83,134.01, 131.43, 122.91, 83.81, 77.85, −3.77.

Dimethyl(4-vinylphenyl)silane (Compound 1n)

In a 100 mL round-bottom flask, 1-bromo-4-vinylbenzene (1.83 g, 10.0mmol) in THF (15 mL) was cooled to −78° C. n-BuLi (7.5 mL, 1.6 M inhexane) was added dropwise very slowly over 30 minutes. The resultingmixture was stirred at −78° C. for 2 hours before the dropwise additionof a solution of chlorodimethylsilane (1.1 mL, 10.0 mmol) in THF (20mL). The reaction was allowed to warm to room temperature and stirredfor 8 hours. The reaction mixture was quenched with NH₄Cl (5 mL, sat.aq.) and the product was extracted with Et₂O (15 mL×3). The organiclayer was washed with water (20 mL), then brine (20 mL), then dried overMgSO₄ and concentrated under reduced pressure (200 torr). The crudeproduct was purified by silica column chromatography with pentane toafford 1n (1.29 g, 7.95 mmol, 80%). This compound is known (136). ¹H NMR(400 MHz, CDCl₃) δ 7.51 (d, J=8.1 Hz, 2H), 7.41 (d, J=7.9 Hz, 2H), 6.72(dd, J=17.6, 10.9 Hz, 1H), 5.79 (dd, J=17.6, 0.9 Hz, 1H), 5.27 (dd,J=10.9, 0.9 Hz, 1H), 4.43 (hept, J=3.7 Hz, 1H), 0.35 (d, J=3.8 Hz, 6H).

Cyclohexa-2,5-dien-1-yldimethylsilane (Compound 1o)

In a 100 mL round-bottom flask, cyclohexa-1,4-diene (2.3 mL, 24.0 mmol)in THF (20 mL) was cooled to −78° C. t-BuLi (15.5 mL, 1.7 M in pentane)and N,N,N′,N′-tetramethylethane-1,2-diamine (TMEDA, 3.6 mL, 24 mmol)were added simultaneously as separate solutions, dropwise over 20minutes. The resulting mixture was allowed to warm to −45° C. in 30 minand maintained at −45° C. for another 1 hour before the dropwiseaddition of chlorodimethylsilane (2.6 mL, 24.0 mmol). The reaction wasallowed to warm to room temperature and stirred for 1.5 hours. Thereaction mixture was quenched with NH₄Cl (5 mL, sat. aq.) and theproduct was extracted with Et₂O (15 mL×3). The organic layer was washedwith water (20 mL), then brine (20 mL), then dried over MgSO₄ andconcentrated under reduced pressure (300 torr). The crude product waspurified by silica column chromatography with pentane to afford Compound1o (1.24 g, 8.97 mmol, 38%). (Note: this compound is oxygen sensitiveand very volatile; storage under argon at −20° C. is recommended.) Thiscompound is known (137). ¹H NMR (400 MHz, CDCl₃) δ 5.72-5.64 (m, 2H),5.61-5.54 (m, 2H), 3.85 (heptd, J=3.5, 1.6 Hz, 1H), 2.80-2.60 (m, 2H),2.42-2.30 (m, 1H), 0.10 (d, J=3.7 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ125.91, 122.09, 29.38, 26.50, −6.31.

Dimethyl(naphthalen-2-yl)silane (Compound 1p)

In a 100 mL round-bottom flask, 2-bromonaphthalene (2.07 g, 10.0 mmol)in THF (15 mL) was cooled to −78° C. n-BuLi (7.5 mL, 1.6 M in hexane)was added dropwise very slowly over 30 minutes. The resulting mixturewas stirred at −78° C. for 2 hours before the dropwise addition of asolution of chlorodimethylsilane (1.1 mL, 10.0 mmol) in THF (20 mL). Thereaction was allowed to warm to room temperature and stirred for 8hours. The reaction mixture was quenched with NH₄Cl (5 mL, sat. aq.) andthe product was extracted with Et₂O (15 mL×3). The organic layer waswashed with water (20 mL), then brine (20 mL), then dried over MgSO₄ andconcentrated under reduced pressure (100 torr). The crude product waspurified by silica column chromatography with pentane to afford Compound1p (1.69 g, 9.07 mmol, 91%). This compound is known (138). ¹H NMR (400MHz, CDCl₃) δ 8.05 (s, 1H), 7.87-7.82 (m, 3H), 7.61 (dd, J=8.1, 1.0 Hz,1H), 7.51-7.48 (m, 2H), 4.56 (hept, J=3.8 Hz, 1H), 0.43 (d, J=3.8 Hz,6H).

Benzofuran-2-yldimethylsilane (Compound 1q)

In a 100 mL round-bottom flask, benzofuran (1.18 g, 10.0 mmol) in THF(15 mL) was cooled to −78° C. n-BuLi (7.5 mL, 1.6 M in hexane) was addeddropwise slowly over 20 minutes. The resulting mixture was allowed towarm to −40° C. within 1 hour and maintained at −40° C. for another 2hours before the dropwise addition of chlorodimethylsilane (1.1 mL, 10.0mmol). The reaction was allowed to warm to room temperature and stirredfor 8 hours. The reaction mixture was quenched with NH₄Cl (5 mL, sat.aq.) and the product was extracted with Et₂O (15 mL×3). The organiclayer was washed with water (20 mL), then brine (20 mL), then dried overMgSO₄ and concentrated under reduced pressure (100 torr). The crudeproduct was purified by silica column chromatography with Et₂O/pentane(1:30) to afford Compound 1q (1.08 g, 6.13 mmol, 61%). This compound isknown (139). ¹H NMR (400 MHz, CDCl₃) δ 7.59 (ddd, J=7.1, 1.3, 0.7 Hz,1H,), 7.52 (dd, J=8.5, 0.5 Hz, 1H), 7.30 (ddd, J=8.5, 7.5, 1.0 Hz, 1H),7.22 (ddd, J=7.5, 7.5, 1.0 Hz, 1H), 7.04 (d, J=0.7 Hz, 1H), 4.52 (hept,J=3.8 Hz, 1H), 0.44 (d, J=3.8 Hz, 6H).

Benzothiophen-2-yldimethylsilane (Compound 1r)

In a 100 mL round-bottom flask, benzothiophene (1.34 g, 10.0 mmol) inTHF (15 mL) was cooled to −78° C. n-BuLi (7.5 mL, 1.6 M in hexane) wasadded dropwise slowly over 20 minutes. The resulting mixture was allowedto warm to −40° C. within 1 hour and maintained at −40° C. for another 2hours before the dropwise addition of chlorodimethylsilane (1.1 mL, 10.0mmol). The reaction was allowed to warm to room temperature and stirredfor 8 hours. The reaction mixture was quenched with NH₄Cl (5 mL, sat.aq.) and the product was extracted with Et₂O (15 mL×3). The organiclayer was washed with water (20 mL), then brine (20 mL), then dried overMgSO₄ and concentrated under reduced pressure (100 torr). The crudeproduct was purified by silica column chromatography with pentane toafford Compound 1r (1.76 g, 9.14 mmol, 91%). ¹H NMR (400 MHz, CDCl₃) δ7.92-7.86 (m, 1H), 7.86-7.79 (m, 1H), 7.53 (s, 1H), 7.38-7.30 (m, 2H),4.63 (hept, J=3.7 Hz, 1H), 0.46 (d, J=3.7 Hz, 6H). ¹³C NMR (100 MHz,CDCl₃) δ 143.87, 141.08, 138.57, 132.18, 124.47, 124.20, 123.63, 122.32,−2.93. FIRMS (FAB) m/z: 191.0354 (M−H⁻); calc. for C₁₀H₁₂SSi: 191.0351.

Synthesis and Characterization of Authentic Organosilicon Products

Racemic standard references of organosilicon products were preparedusing rhodium-catalyzed Si—H insertion reactions, following the generalprocedure described below as follows.

General Procedure for Rhodium-Catalyzed Si—H insertion

To a 20 mL vial or 25 mL flask was added silane (1.0 mmol, 1.0 equiv.),Rh₂(OAc)₄ (4.4 mg, 1 mol %) and DCM (5 mL). The mixture was cooled to−78° C., after which diazo compound (1.0 mmol, 1.0 equiv.) was addeddropwise to the solution. The reaction was allowed to slowly warm up toroom temperature in 8 hours and stirred at room temperature for another4 hours. Evaporation of the organic solvent and purification by silicacolumn chromatography using EtOAc and hexane as eluents affordedorganosilicon Compounds 3-20 in 20-70% yields.

Organosilicon compounds 21 and 22 were prepared by rhodium-catalyzedSi—H insertion between Me-EDA and the corresponding 0- or N-protectedsilane (Compounds 1ks and 23s, respectively) to give Compounds 21a and22a, followed by deprotection under standard palladium-catalyzedhydrogenation condition (10% Pd/C in ethanol under H₂ atmosphere at roomtemperature for 16 hours). The hydrogenation reaction afforded Compounds21 and 22 in 96% and 88% yield, respectively. The chemical structuresand NMR data for Compounds 3-20, 21a, 21, 22, and 22 are shown below:

Ethyl 2-(dimethyl(phenyl)silyl)propanoate (Compound 3)

¹H NMR (400 MHz, CDCl₃) δ 7.55-7.46 (m, 2H), 7.43-7.32 (m, 3H), 4.02 (q,J=7.1 Hz, 2H), 2.25 (q, J=7.1 Hz, 1H), 1.17-1.11 (m, 6H), 0.37 (d, J=0.6Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 176.13, 136.42, 133.99, 129.57,127.92, 59.96, 30.14, 14.43, 11.42, −3.92, −4.77. HRMS (FAB) m/z:236.1234 (M⁻); calc. for C₁₃H₂₀O₂Si: 236.1233.

Ethyl 2-(dimethyl(p-tolyl)silyl)propanoate (Compound 4)

¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J=7.9 Hz, 2H), 7.18 (d, J=7.5 Hz,2H), 4.03 (q, J=7.1 Hz, 2H), 2.35 (s, 3H), 2.24 (q, J=7.1 Hz, 1H),1.19-1.11 (m, 6H), 0.35 (d, J=0.4 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ176.21, 139.51, 134.04, 132.72, 128.77, 59.94, 30.22, 21.63, 14.47,11.44, −3.75, −4.75. ²⁹Si NMR (79 MHz, CDCl₃) δ 0.19. FIRMS (FAB) m/z:250.1391 (M⁺); calc. for C₁₄H₂₂O₂Si: 250.1389.

Ethyl 2-(dimethyl(4-methoxyphenyl)silyl)propanoate (Compound 5)

¹H NMR (400 MHz, CDCl₃) δ 7.43 (d, J=8.6 Hz, 2H), 6.91 (d, J=8.6 Hz,2H), 4.03 (q, J=7.1 Hz, 2H), 3.81 (s, 3H), 2.22 (q, J=7.1 Hz, 1H),1.20-1.10 (m, 6H), 0.35 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 176.24,160.84, 135.49, 127.18, 113.70, 59.93, 55.18, 30.37, 14.49, 11.45,−3.72, −4.58. ²⁹Si NMR (79 MHz, CDCl₃) δ −0.10. HRMS (FAB) m/z: 266.1332(M⁺); calc. for C₁₄H₂₂O₃Si: 266.1338.

Ethyl 2-(dimethyl(4-chlorophenyl)silyl)propanoate (Compound 6)

¹H NMR (400 MHz, CDCl₃) δ 7.43 (d, J=8.4 Hz, 2H), 7.34 (d, J=8.3, 1.9Hz, 2H), 4.02 (q, J=7.1 Hz, 2H), 2.23 (q, J=7.1 Hz, 1H), 1.17-1.11 (m,6H), 0.36 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 175.87, 135.98, 135.36,134.75, 128.19, 60.06, 30.03, 14.45, 11.38, −4.02, −4.61. ²⁹Si NMR (79MHz, CDCl₃) δ 0.59. HRMS (FAB) m/z: 270.0847 (M⁺); calc. forC₁₃H₁₉O₂SiCl: 270.0843.

Ethyl 2-(dimethyl(4-trifluoromethylphenyl)silyl)propanoate (Compound 7)

¹H NMR (400 MHz, CDCl₃) δ 7.61 (d, J=8.4 Hz, 4H), 4.01 (qd, J=7.2, 0.7Hz, 2H), 2.27 (q, J=7.2 Hz, 1H), 1.16 (d, J=7.2 Hz, 3H), 1.12 (t, J=7.1Hz, 3H), 0.40 (d, J=1.3 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 141.49,134.32, 131.52 (q, J_(C-F)=32.3 Hz), 124.26 (q, J_(C-F)=272.2 Hz),124.43 (q, J_(C-F)=3.8 Hz), 60.16, 29.96, 14.39, 11.39, −4.18, −4.65.²⁹Si NMR (79 MHz, CDCl₃) δ 1.01. HRMS (FAB) m/z: 304.1119 (M⁺); calc.for C₁₄H₁₉O₂SiF₃: 304.1106.

Ethyl 2-(dimethyl(4-chloromethylphenyl)silyl)propanoate (Compound 8)

¹H NMR (400 MHz, CDCl₃) δ 7.51 (d, J=8.0 Hz, 2H), 7.38 (d, J=7.9 Hz,2H), 4.58 (s, 2H), 4.02 (q, J=7.1 Hz, 2H), 2.25 (q, J=7.1 Hz, 1H),1.18-1.08 (m, 6H), 0.37 (d, J=1.2 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ175.98, 138.72, 136.92, 134.44, 128.02, 60.02, 46.23, 30.05, 14.44,11.41, −3.97, −4.70. ²⁹Si NMR (79 MHz, CDCl₃) δ 0.59. FIRMS (FAB) m/z:283.0927 ((M+H)—H₂ ⁺); calc. for C₁₄H₂₀O₂SiCl: 283.0921.

Methyl 4-((1-ethoxy-1-oxopropan-2-yl)dimethylsilyl)benzoate (Compound 9)

¹H NMR (400 MHz, CDCl₃) δ 8.00 (d, J=8.3 Hz, 2H), 7.58 (d, J=8.3 Hz,2H), 4.01 (qd, J=7.1, 0.9 Hz, 2H), 3.92 (s, 3H), 2.27 (q, J=7.2 Hz, 1H),1.19-1.05 (m, 6H), 0.39 (d, J=1.4 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ175.77, 167.27, 142.78, 134.01, 131.00, 128.64, 60.07, 52.31, 29.88,14.44, 11.37, −4.13, −4.71. ²⁹Si NMR (79 MHz, CDCl₃) δ 0.98. HRMS (FAB)m/z: 294.1276 (M⁺); calc. for C₁₅H₂₂O₄Si: 294.1287.

Ethyl 2-((4-(dimethylcarbamoyl)phenyl)dimethylsilyl)propanoate (Compound10)

¹H NMR (400 MHz, CDCl₃) δ 7.54 (d, J=8.0 Hz, 2H), 7.39 (d, J=8.0 Hz,2H), 4.03 (q, J=7.1 Hz, 2H), 3.11 (s, 3H), 2.97 (s, 3H), 2.26 (q, J=7.2Hz, 1H), 1.20-1.10 (m, 6H), 0.37 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ175.94, 171.59, 138.39, 137.38, 134.01, 126.37, 60.05, 39.67, 35.45,29.95, 14.49, 11.41, −3.92, −4.75. FIRMS (FAB) m/z: 308.1689 (M+H⁺);calc. for C₁₆H₂₆NO₃Si: 308.1682.

Ethyl 2-((naphthalen-2-yl)dimethylsilyl)propanoate (Compound 11)

¹H NMR (400 MHz, CDCl₃) δ 8.00 (s, 1H), 7.87-7.80 (m, 3H), 7.57 (dd,J=8.2, 0.8 Hz, 1H), 7.53-7.46 (m, 2H), 4.02 (q, J=7.1 Hz, 2H), 2.34 (q,J=7.1 Hz, 1H), 1.18 (d, J=7.1 Hz, 3H), 1.11 (t, J=7.1 Hz, 3H), 0.46 (d,J=2.0 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 176.14, 134.88, 133.99,133.92, 132.92, 130.03, 128.23, 127.84, 127.16, 126.70, 126.15, 60.01,30.18, 14.44, 11.50, −3.76, −4.71. ²⁹Si NMR (79 MHz, CDCl₃) δ 0.76. HRMS(FAB) m/z: 286.1395 (M⁺); calc. for C₁₇H₂₂O₂Si: 286.1389.

Ethyl 2-((benzofuran-2-yl)dimethylsilyl)propanoate (Compound 12)

¹H NMR (400 MHz, CDCl₃) δ 7.58 (ddd, J=7.7, 1.3, 0.7 Hz, 1H), 7.50 (dd,J=8.2, 0.9 Hz, 1H), 7.29 (ddd, J=8.4, 7.2, 1.4 Hz, 1H), 7.21 (ddd,J=8.2, 7.8, 1.0 Hz, 1H), 7.05 (d, J=1.0 Hz, 1H), 4.07 (qd, J=7.2, 1.2Hz, 2H), 2.38 (q, J=7.2 Hz, 1H), 1.24 (d, J=7.1 Hz, 3H), 1.14 (t, J=7.1Hz, 3H), 0.44 (d, J=1.9 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 175.57,159.85, 158.31, 127.82, 124.84, 122.58, 121.29, 117.93, 111.48, 60.22,29.42, 14.42, 11.24, −4.32, −5.05. ²⁹Si NMR (79 MHz, CDCl₃) δ −5.09.HRMS (FAB) m/z: 276.1182 (M⁺); calc. for C₁₅H₂₀O₃Si: 276.1169.

Ethyl 2-(benzothiophen-2-yldimethylsilyl)propanoate (Compound 13)

¹H NMR (400 MHz, CDCl₃) δ 7.93-7.85 (m, 1H), 7.85-7.79 (m, 1H), 7.52 (d,J=0.8 Hz, 1H), 7.39-7.30 (m, 2H), 4.08 (qd, J=7.1, 1.1 Hz, 2H), 2.33 (q,J=7.2 Hz, 1H), 1.24 (d, J=7.2 Hz, 3H), 1.17 (t, J=7.1 Hz, 3H), 0.47 (d,J=0.9 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 175.61, 143.84, 140.93,137.78, 132.56, 124.64, 124.25, 123.75, 122.30, 60.24, 30.46, 14.47,11.43, −2.77, −3.64. ²⁹Si NMR (79 MHz, CDCl₃) δ −1.51. HRMS (FAB) m/z:292.0963 (M⁺); calc. for C₁₅H₂₀O₂SiS: 292.0953.

Ethyl 2-(benzyldimethylsilyl)propanoate (Compound 14)

¹H NMR (400 MHz, CDCl₃) δ 7.25-7.17 (m, 2H), 7.11-7.06 (m, 1H),7.06-6.96 (m, 2H), 4.24-4.02 (m, 2H), 2.24-2.14 (m, 2H), 2.10 (q, J=7.1Hz, 1H), 1.26 (t, J=7.1 Hz, 3H), 1.19 (d, J=7.1 Hz, 3H), 0.06-−0.12 (m,J=0.5 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 176.19, 139.25, 128.44,128.42, 124.44, 60.04, 28.95, 24.03, 14.66, 11.17, −4.76, −4.82. ²⁹SiNMR (79 MHz, CDCl₃) δ 5.96. FIRMS (FAB) m/z: 251.1459 (M+H⁺); calc. forC₁₄H23O₂Si: 251.1467.

Ethyl 2-(cyclohexa-2,5-dien-1-yldimethylsilyl)propanoate (Compound 15)

¹H NMR (400 MHz, CDCl₃) δ 5.81-5.52 (m, 4H), 4.17-4.05 (m, 2H),2.81-2.56 (m, 2H), 2.48-2.36 (m, 1H), 2.19 (q, J=7.1 Hz, 1H), 1.24 (t,J=7.1 Hz, 3H), 1.21 (d, J=7.2 Hz, 3H), 0.08 (d, J=4.4 Hz, 6H). ¹³C NMR(100 MHz, CDCl₃) δ 176.24, 125.63, 125.53, 122.65, 122.52, 60.05, 29.47,28.28, 26.52, 14.59, 11.33, −6.35, −6.70. HRMS (FAB) m/z: 237.1313((M+H)—H₂ ⁺); calc. for C₁₃H₂₁O₂Si: 237.1311.

Ethyl 2-((4-ethynylphenyl)dimethylsilyl)butanoate (Compound 16)

¹H NMR (400 MHz, CDCl₃) δ 7.53-7.42 (m, 4H), 4.13-3.93 (m, 2H), 3.11 (s,1H), 2.08 (dd, J=11.7, 3.1 Hz, 1H), 1.78 (ddq, J=14.2, 11.7, 7.1 Hz,1H), 1.40 (dqd, J=13.8, 7.3, 3.1 Hz, 1H), 1.13 (t, J=7.1 Hz, 3H), 0.89(t, J=7.2 Hz, 3H), 0.35 (d, J=4.1 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ175.05, 137.86, 133.87, 131.36, 123.17, 83.71, 78.06, 59.95, 39.53,20.60, 15.13, 14.47, −3.96, −4.56. HRMS (FAB) m/z: 273.1316 ((M+H)—H₂⁺); calc. for C₁₆H₂₁O₂Si: 273.1311.

Isopropyl 2-((4-ethynylphenyl)dimethylsilyl)propanoate (Compound 17)

¹H NMR (400 MHz, CDCl₃) δ 7.47 (app s, 4H), 4.91 (hept, J=6.3 Hz, 1H),3.11 (s, 1H), 2.21 (q, J=7.1 Hz, 1H), 1.16 (d, J=6.3 Hz, 3H), 1.13 (d,J=7.2 Hz, 3H), 1.08 (d, J=6.3 Hz, 3H), 0.36 (d, J=3.0 Hz, 6H). ¹³C NMR(100 MHz, CDCl₃) δ 175.39, 137.82, 133.93, 131.36, 123.15, 83.73, 78.03,67.33, 30.02, 22.16, 22.01, 11.45, −3.95, −4.69. HRMS (FAB) m/z:275.1479 (M+H⁺); calc. for C₁₆H₂₃O₂Si: 275.1467.

Ethyl 2-((3,4-dihydro-2H-pyran-6-yl)dimethylsilyl)propanoate (Compound18)

¹H NMR (400 MHz, CDCl₃) δ 5.02 (t, J=3.8, 1H), 4.16-4.02 (m, 2H), 3.90(dd, 4.9 Hz, 2H), 2.17 (q, J=7.1 Hz, 1H), 2.05-1.98 (m, 2H), 1.87-1.78(m, 2H), 1.23 (t, J=7.1 Hz, 3H), 1.18 (d, J=7.1 Hz, 3H), 0.14 (d, J=8.6Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 176.26, 157.26, 112.83, 65.77,59.91, 28.78, 22.79, 20.90, 14.61, 11.24, −4.96, −5.98. ²⁹Si NMR (79MHz, CDCl₃) δ −4.02. HRMS (FAB) m/z: 243.1410 (M+H⁺); calc. forC₁₂H₂₃O₃Si: 243.1417.

Ethyl 2-((4-vinylphenyl)dimethylsilyl)propanoate (Compound 19)

¹H NMR (400 MHz, CDCl₃) δ 7.47 (d, J=8.1 Hz, 2H), 7.40 (d, J=8.0 Hz,2H), 6.71 (dd, J=17.6, 10.9 Hz, 1H), 5.79 (dd, J=17.6, 0.9 Hz, 1H), 5.27(dd, J=10.9, 1.0 Hz, 1H), 4.03 (q, J=7.1 Hz, 2H), 2.25 (q, J=7.1 Hz,1H), 1.16-1.13 (m, 6H), 0.37 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 176.09,138.68, 136.85, 136.00, 134.26, 125.69, 114.65, 59.99, 30.14, 14.46,11.42, −3.87, −4.75. ²⁹Si NMR (79 MHz, CDCl₃) δ 0.26. FIRMS (FAB) m/z:262.1384 (M⁺); calc. for C₁₅H₂₂O₂Si: 262.1389.

Ethyl 2-((4-ethynylphenyl)dimethylsilyl)propanoate (Compound 20)

¹H NMR (400 MHz, CDCl₃) δ 7.50-7.44 (m, 4H), 4.01 (qd, J=7.2, 0.6 Hz,2H), 3.11 (s, 1H), 2.24 (q, J=7.1 Hz, 1H), 1.17-1.10 (m, 6H), 0.37 (d,J=0.7 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 175.89, 137.69, 133.87,131.36, 123.20, 83.70, 78.08, 60.04, 29.98, 14.44, 11.38, −4.10, −4.75.²⁹Si NMR (79 MHz, CDCl₃) δ 0.79. HRMS (FAB) m/z: 259.1153 ((M+H)—H₂ ⁺);calc. for C₁₅H₁₉O₂Si: 259.1154.

Ethyl 2-((4-(benzyloxy)phenyl)dimethylsilyl)propanoate (Compound 21a)

¹H NMR (400 MHz, CDCl₃) δ 7.46-7.30 (m, 7H), 7.05-6.93 (m, 2H), 5.08 (s,2H), 4.02 (q, J=7.1 Hz, 2H), 2.22 (q, J=7.1 Hz, 1H), 1.20-1.10 (m, 6H),0.35 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 176.22, 160.07, 137.01, 135.51,128.74, 128.13, 127.61, 127.52, 114.56, 69.87, 59.93, 30.35, 14.48,11.45, −3.75, −4.57. HRMS (FAB) m/z: 342.1665 (M⁺); calc. forC₂₀H₂₆O₃Si: 342.1651.

Ethyl 2-((4-hydroxyphenyl)dimethylsilyl)propanoate (Compound 21)

¹H NMR (400 MHz, CDCl₃) δ 7.36 (d, J=8.5 Hz, 2H), 6.81 (d, J=8.6 Hz,2H), 5.49 (s, 1H), 4.03 (qd, J=7.1, 0.6 Hz, 2H), 2.23 (q, J=7.1 Hz, 1H),1.19-1.12 (m, 6H), 0.35 (d, J=1.2 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ176.67, 157.18, 135.68, 126.90, 115.16, 60.12, 30.51, 14.46, 11.43,−3.83, −4.33. HRMS (FAB) m/z: 252.1175 (M⁺); calc. for C₁₃H₂₀O₃Si:252.1182.

Ethyl 2-((4-(((benzyloxy)carbonyl)amino)phenyl)dimethylsilyl)propanoate(Compound 22a)

¹H NMR (400 MHz, CDCl₃) δ 7.49-7.30 (m, 9H), 6.70 (s, 1H), 5.20 (s, 2H),4.02 (q, J=7.1 Hz, 2H), 2.22 (q, J=7.1 Hz, 1H), 1.18-1.10 (m, 6H), 0.35(s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 176.13, 153.24, 139.07, 136.06,134.98, 130.78, 128.78, 128.56, 128.48, 117.96, 67.24, 59.98, 30.21,14.49, 11.41, −3.83, −4.68. HRMS (FAB) m/z: 385.1710 (M⁺); calc. forC₂₁H₂₇NO₄Si: 385.1709.

Ethyl 2-((4-aminophenyl)dimethylsilyl)propanoate (Compound 22)

¹H NMR (400 MHz, CDCl₃) δ 7.29 (d, J=8.4 Hz, 2H), 6.70 (d, J=8.4 Hz,2H), 4.03 (q, J=7.1 Hz, 2H), 3.64 (br s, 2H), 2.20 (q, J=7.1 Hz, 1H),1.16 (t, J=7.1 Hz, 3H), 1.13 (d, J=7.1 Hz, 3H), 0.32 (s, 6H). ¹³C NMR(100 MHz, CDCl₃) δ 176.40, 147.40, 135.32, 124.37, 114.82, 59.87, 30.47,14.48, 11.44, −3.68, −4.68. HRMS (FAB) m/z: 251.1339 (M⁺); calc. forC₁₃H₂₁NO₂Si: 251.1342. [αD]²⁵=+46.1 (c 0.505 in cyclohexane, 98% ee).

Preparative-Scale Whole-Cell Biocatalytic Reaction

FIG. 14 shows a preparative-scale whole-cell biocatalytic reaction.HB_(amp/chlor) (50 mL) in a 250 mL flask was inoculated with anovernight culture (1 mL, LB_(amp/chlor)) of recombinant E. cloni®EXPRESS BL21(DE3) cells containing a pET22(b)+ plasmid encoding Rma cytc V75T M100D M103D, and the pEC86 plasmid. The culture was shaken at 37°C. and 250 rpm (no humidity control) until the OD₆₀₀ was 0.6(approximately 2 hours). The culture was placed on ice for 30 minutes,and IPTG and 5-ALA were added to final concentrations of 20 μM and 200μM, respectively. The incubator temperature was reduced to 20° C., andthe culture was allowed to shake for 20 hours at 140 rpm. Cells werepelleted by centrifugation (4° C., 5 minutes, 4,000×g), resuspended inM9-N buffer, and adjusted to OD₆₀₀=15. Under anaerobic conditions, to a5 mL reaction vial was added 960 μL whole-cell solution, 40 μL glucosesolution (250 mM in M9-N buffer), 15.8 μL 4-(dimethylsilyl)aniline(Compound 23, 0.1 mmol), and 4.9 μL Me-EDA (0.04 mmol) at roomtemperature. The reaction was shaken at 480 rpm. At 4 h intervals, twoadditional batches of whole cell (960 μL), glucose (40 μL) and Me-EDA(4.9 μL, 0.04 mmol) were added to the reaction. After the reaction wasshaken for a total of 20 hours, the reaction mixture was divided betweenfour 2 mL microcentrifuge tubes, and 0.6 mL EtOAc was added to eachtube. The reaction mixtures were subjected to vortex (30 seconds) andcentrifugation (14,000 rpm, 7 min) to completely separate the organicand aqueous layers. After removal of the organic layers, two additionrounds of extraction were carried out. The combined organic layers weredried over anhydrous Na₂SO₄, concentrated, and purified by silica columnchromatography with EtOAc/hexane (1:4 to 1:2) to afford pureorganosilicon product Compound 22 (17.6 mg, 0.0700 mmol, 70% yield),together with recovered silane 23 (4.0 mg, 0.0265 mmol, 26.5%). Thestereoselectivity of the product was determined as 98% ee by chiral SFC.The protein concentration of OD₆₀₀=15 whole-cell solution was determinedto be 7.43 μM by the ferrous assay after cell lysis by sonication. Thetotal turnover number for this reaction was 3,410.

GC Standard Curves for Organosilicon Products

The analysis of product formation in enzymatic reactions was performedbased on gas chromatography (GC) standard curves. The general procedurefor preparing analytical samples for GC standard curves is describedbelow.

Sample Preparation for GC Standard Curves

Stock solutions of chemically synthesized organosilicon products atvarious concentrations (20 to 200 mM in MeCN) were prepared. To amicrocentrifuge tube was added 340 μL M9-N buffer, 40 μL Na₂S₂O₄ (100 mMin dH₂O), 20 μL organosilicon product, 20 μL internal standard (20 mM2-phenylethanol in cyclohexane), and 1 mL cyclohexane. The mixture wasvortexed (10 seconds, 3 times) then centrifuged (14,000×g, 5 minutes) tocompletely separate the organic and aqueous layers. The organic layer(750 μL) was removed for GC analysis.

Chiral Supercritical Fluid Chromatography (SFC) Analysis of Racemic andEnzymatically Synthesized Organosilicon Products

All the ee values of enzymatically synthesized organosilicon productswere determined using chiral SFC. The chiral SFC reports for racemic andenzymatic products are shown below. The absolute configuration ofenzymatically synthesized organosilicon Compound 3 was determined to be(R) by comparing the HPLC retention times of Compound rac-3 and Compound3 with that reported in the literature (84). The absolute configurationsof other organosilicon products were inferred by analogy, assuming thefacial selectivity of the diazo reagents remained the same in thebiosyntheses of Compounds 3-22.

rac or R-3

TABLE 9 Area % report for rac and R-3 (Chiralpak IC, 3% i-PrOH in CO₂,3.5 mL/min, 210 nm) rac-3 R-3 Retention Area Retention Area Time (min)(mAU * s) Area % Time (min) (mAU * s) Area % 2.224 842.723 50.03 2.172753.160 100.00 2.390 841.657 49.97 — — Total 1684.380 100.00 Total753.160 100.00

rac or R-4

TABLE 10 Area % report for rac and R-4 (Chiralpak AD-H, 3% i-PrOH inCO₂, 2.5 mL/min, 210 nm) rac-4 R-4 Retention Area Retention Area Time(min) (mAU * s) Area % Time (min) (mAU * s) Area % 2.097 124486 45.442.142 16934.0 100.00 2.390 149493 54.56 — — Total 273979 100.00 Total16934.0 100.00

rac or R-5

TABLE 11 Area % report for rac and R-5 (Chiralpak IC, 3% i-PrOH in CO₂,3.5 mL/min, 210 nm) rac-5 R-5 Retention Area Retention Area Time (min)(mAU * s) Area % Time (min) (mAU * s) Area % 3.869 8651.07 49.14 3.8731422.48 100.00 4.349 8955.03 50.86 — — Total 17606.10 100.00 Total1422.48 100.00

rac or R-6

TABLE 12 Area % report for rac and R-6(Chiralpak AD-H, 3% i-PrOH in CO₂,2.5 mL/min, 210 nm) rac-6 R-6 Retention Area Retention Area Time (min)(mAU * s) Area % Time (min) (mAU * s) Area % 2.510 4898.62 49.48 2.477455.973 100.00 2.746 5001.36 50.52 — — Total 9899.98 100.00 Total455.973 100.00

rac or R-7

TABLE 13 Area % report for rac and R-7 (Chiralcel OD-H, 2% i-PrOH inCO₂, 2.5 mL/min, 210 nm) rac-7 R-7 Retention Area Retention Area Time(min) (mAU * s) Area % Time (min) (mAU * s) Area % 2.186 3895.65 49.542.182 1738.84 99.61 2.431 3967.58 50.46 2.402 6.80 0.39 Total 7863.23100.00 Total 1745.65 100.00

rac or R-8

TABLE 14 Area % report for rac and R-8 (Chiralpak AD-H, 3% i-PrOH inCO₂, 2.5 mL/min, 210 nm) rac-8 R-8 Retention Area Retention Area Time(min) (mAU * s) Area % Time (min) (mAU * s) Area % 3.406 1698.74 49.773.409 2377.97 99.58 3.914 1714.41 50.23 3.912 10.15 0.42 Total 3413.15100.00 Total 2388.12 100.00

rac or R-9

TABLE 15 Area % report for rac and R-9 (Chiralcel OD-H, 5% i-PrOH inCO₂, 2.5 mL/min, 210 nm) rac-9 R-9 Retention Area Retention Area Time(min) (mAU * s) Area % Time (min) (mAU * s) Area % 3.517 1773.21 50.003.507 582.064 100.00 3.824 1772.88 50.00 — — Total 3546.09 100.00 Total582.064 100.00

rac or R-10

TABLE 16 Area % report for rac and R-10 (Chiralpak IC, 30% i-PrOH inCO₂, 2.5 mL/min, 210 nm) rac-10 R-10 Retention Area Retention Area Time(min) (mAU * s) Area % Time (min) (mAU * s) Area % 6.693 2779.93 49.386.665 987.666 99.63 7.000 2849.58 50.62 7.005 3.594 0.36 Total 5629.50100.00 Total 991.260 100.00

rac or R-11

TABLE 17 Area % report for rac and R-11 (Chiralpak IC, 7% i-PrOH in CO₂,3.5 mL/min, 210 nm) rac-11 R-11 Retention Area Retention Area Time (min)(mAU * s) Area % Time (min) (mAU * s) Area % 2.735 8677.04 47.45 2.7603675.15 97.51 3.183 9609.04 52.55 3.215 93.95 2.49 Total 18286.1 100.00Total 3769.10 100.00

rac or R-12

TABLE 18 Area % report for rac and R-12 (Chiralpak IC, 3% i-PrOH in CO₂,3.5 mL/min, 210 nm) rac-12 R-12 Retention Area Retention Area Time (min)(mAU * s) Area % Time (min) (mAU * s) Area % 3.101 7145.58 49.41 3.0341490.62 99.16 3.624 7315.99 50.59 3.549 12.69 0.84 Total 14461.6 100.00Total 1503.31 100.00

rac or R-13

TABLE 19 Area % report for rac and R-13 (Chiralpak IC, 7% i-PrOH in CO₂,3.5 mL/min, 210 nm) rac-13 R-13 Retention Area Retention Area Time (min)(mAU * s) Area % Time (min) (mAU * s) Area % 2.751 7136.78 48.49 2.764801.009 98.78 3.209 7581.08 51.51 3.219 9.889 1.22 Total 14717.9 100.00Total 810.898 100.00

rac or R-14

TABLE 20 Area % report for rac and R-14 (Chiralpak AD-H, 3% i-PrOH inCO₂, 2.5 mL/min, 210 nm) rac-14 R-14 Retention Area Retention Area Time(min) (mAU * s) Area % Time (min) (mAU * s) Area % 2.142 1384.73 49.852.191 1991.26 99.62 2.351 1393.07 50.15 2.402 7.58 0.38 Total 2777.80100.00 Total 1998.84 100.00

rac or R-15

TABLE 21 Area % report for rac and R-15 (Chiralpak IC, 3% i-PrOH in CO₂,3.5 mL/min, 210 nm) rac-15 R-15 Retention Area Retention Area Time (min)(mAU * s) Area % Time (min) (mAU * s) Area % 2.356 361.685 50.30 2.426390.115 99.45 2.500 357.384 49.70 2.556 2.125 0.54 Total 719.070 100.00Total 392.240 100.00

rac or R-16

TABLE 22 Area % report for rac and R-16 (Chiralpak AD-H, 3% i-PrOH inCO₂, 2.5 mL/min, 210 nm) rac-16 R-16 Retention Area Retention Area Time(min) (mAU * s) Area % Time (min) (mAU * s) Area % 2.602 4276.04 49.442.607 2426.65 100.00 3.526 4373.23 50.56 Total 8649.28 100.00 Total2426.65 100.00

rac or R-17

TABLE 23 Area % report for rac and R-17 (Chiralpak IC, 3% i-PrOH in CO₂,2.5 mL/min, 210 nm) rac-17 R-17 Retention Area Retention Area Time (min)(mAU * s) Area % Time (min) (mAU * s) Area % 3.808 1442.74 49.78 3.8292089.15 100.00 4.074 1455.49 50.22 Total 2898.23 100.00 Total 2089.15100.00

rac or R-18

TABLE 24 Area % report for rac and R-18 (Chiralpak IC, 3% i-PrOH in CO₂,3.5 mL/min, 210 nm) rac-18 R-18 Retention Area Retention Area Time (min)(mAU * s) Area % Time (min) (mAU * s) Area % 2.680 1366.02 50.54 2.628813.657 99.63 2.887 1336.57 49.46 2.922 2.984 0.37 Total 2702.59 100.00Total 816.641 100.00

rac or R-19

TABLE 25 Area % report for rac and R-19 (Chiralpak IC, 3% i-PrOH in CO₂,3.5 mL/min, 210 nm) rac-19 R-19 Retention Area Retention Area Time (min)(mAU * s) Area % Time (min) (mAU * s) Area % 2.791 6332.09 49.69 2.7581559.90 99.13 3.001 6411.17 50.31 2.972 13.65 0.87 Total 12743.3 100.00Total 1573.56 100.00

rac or R-20

TABLE 26 Area % report for rac and R-20 (Chiralcel OD-H, 3% i-PrOH inCO₂, 2.5 mL/min, 210 nm) rac-20 R-20 Retention Area Retention Area Time(min) (mAU * s) Area % Time (min) (mAU * s) Area % 3.410 13436.2 48.403.406 1894.53 99.84 3.774 14326.4 51.60 3.715 2.97 0.16 Total 27762.6100.00 Total 1897.50 100.00

rac or R-21

TABLE 27 Area % report for rac and R-21 (Chiralpak IC, 10% i-PrOH inCO₂, 2.5 mL/min, 210 nm) rac-21 R-21 Retention Area Retention Area Time(min) (mAU * s) Area % Time (min) (mAU * s) Area % 3.853 664.781 50.193.883 3332.68 99.58 4.264 659.660 49.81 4.331 13.94 0.42 Total 1324.441100.00 Total 3346.62 100.00

rac or R-22

TABLE 28 Area % report for rac and R-22 (Chiralpak AD-H, 10% i-PrOH inCO₂, 2.5 mL/min, 210 nm) rac-22 R-22 Retention Area Retention Area Time(min) (mAU * s) Area % Time (min) (mAU * s) Area % 5.162 9214.41 48.50 —— 5.617 9783.58 51.50 5.638 195.47 100.00 Total 18998.0 100.00 Total195.47 100.00Preparative-Scale Reaction (0.1 mmol):

TABLE 29 Area % report for R-22: R-22 (OD 15-whole cell) Retention Time(min) Area (mAU * s) Area % 5.537 45.97 1.15 5.990 3935.62 98.85 Total3981.59 100.00

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

INFORMAL SEQUENCE LISTING

SEQ ID NO: Sequence Notes 1 TESGTAAQDPEALAAEIGPVKQVSLGEQIDAALAQQGEQLFNRhodothermus marinus TYCTACHRLDERFIGPALRDVTKRRGPVYIMNVMLNPNGMIQcytochrome c RHPVMKQLVQEYGTMMTDMALSEEQARAILEYLRQVAENQ protein (mature) 2MLLLSLTLAACGGGSSSSTPQPSGSAAQTESGTAAQDPEALA Rhodothermus marinusAEIGPVKQVSLGEQIDAALAQQGEQLFNTYCTACHRLDERFI cytochrome cGPALRDVTKRRGPVYIMNVMLNPNGMIQRHPVMKQLVQEYGT protein (unprocessed)MMTDMALSEEQARAILEYLRQVAENQ 3 MKYLLPTAAAGLLLLAAQPAMA N-terminal pelBleader sequence

What is claimed is:
 1. A method for producing an organosilicon product,the method comprising combining: (a) a silicon-containing reagent,wherein the silicon-containing reagent is a compound of Formula I:

(b) a carbene precursor, wherein the carbene precursor is a diazosubstrate of Formula II:

and (c) a cytochrome c protein variant having at least 90% identity tothe amino acid sequence set forth in SEQ ID NO:1 and one or moremutations selected from the group consisting of V75, M100, and M103relative to the amino acid sequence set forth in SEQ ID NO:1 underconditions sufficient to produce an organosilicon product, wherein theorganosilicon product is a compound of Formula III:

and wherein: R¹, R², R³, and R⁴ are independently selected from thegroup consisting of H, optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to10-membered heteroaryl, optionally substituted 6- to 10-memberedheterocyclyl, cyano, halo, hydroxy, alkoxy, SR⁷, N(R⁸)₂, B(R⁹)₂,Si(R⁹)₃, P(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, andC(O)NR⁷OR⁸; R⁵ and R⁶ are independently selected from the groupconsisting of H, optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₁₋₁₈ haloalkyl, optionally substituted C₂₋₁₈ alkenyl,optionally substituted C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl,optionally substituted 6- to 10-membered heteroaryl, optionallysubstituted 6- to 10-membered heterocyclyl, cyano, halo, N(R⁸)₂, B(R⁹)₂,Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸,C(O)C(O)OR⁷, and P(O)(OR⁷)₂; and each R⁷, R⁸, and R⁹ is independentlyselected from the group consisting of H, optionally substituted C₁₋₁₈alkyl, optionally substituted C₂₋₁₈ alkenyl, optionally substitutedC₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted6- to 10-membered heteroaryl, and optionally substituted 6- to10-membered heterocyclyl.
 2. The method of claim 1, wherein thecytochrome c protein variant can enantioselectively catalyze theformation of a carbon-silicon bond.
 3. The method of claim 1, whereinthe cytochrome c protein variant comprises a heme cofactor that is anon-native cofactor.
 4. The method of claim 1, wherein the cytochrome cprotein variant has a higher total turnover number (TTN) compared to awild-type cytochrome c heme protein.
 5. The method of claim 1, whereinthe cytochrome c protein variant has a higher turnover frequency (TOF)compared to a wild-type cytochrome c protein.
 6. The method of claim 1,wherein the cytochrome c protein variant produces an organosiliconproduct with a % ee of at least about 75%.
 7. The method of claim 1,wherein at least one of R¹, R², R³ and R⁴ is selected from the groupconsisting of optionally substituted C₁₋₁₈ alkyl, optionally substitutedC₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl, optionallysubstituted C₆₋₁₀ aryl, optionally substituted 6- to 10-memberedheteroaryl, optionally substituted 6- to 10-membered heterocyclyl,cyano, halo, hydroxy, alkoxy, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, P(R⁹)₃, C(O)OR⁷,C(O)SR⁷, C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂ and C(O)NR⁷OR⁸.
 8. The methodof claim 1, wherein at least one of R¹, R², R³ and R⁴ is selected fromthe group consisting of optionally substituted C₁₋₁₈ alkyl, optionallysubstituted C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl,optionally substituted C₆₋₁₀ aryl, optionally substituted C₆₋₁₀aryl-C₁₋₆ alkyl, optionally substituted 6- to 10-membered heteroaryl,and optionally substituted 6- to 10-membered heterocyclyl.
 9. The methodof claim 1, wherein R¹ is H and R², R³, and R⁴ are independentlyselected from the group consisting of H, optionally substituted C₁₋₁₈alkyl, optionally substituted C₂₋₁₈ alkenyl, optionally substitutedC₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substitutedC₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6- to 10-memberedheteroaryl, and optionally substituted 6- to 10-membered heterocyclyl,provided that at least one of R², R³, and R⁴ is other than H.
 10. Themethod of claim 1, wherein R² is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl.
 11. The method of claim 1, wherein R³ and R⁴ are C₁₋₆alkyl.
 12. The method of claim 1, wherein R⁵ is C(O)OR⁷.
 13. The methodof claim 1, wherein R⁶ is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈haloalkyl, and optionally substituted C₂₋₁₈ alkenyl.
 14. The method ofclaim 1, wherein: R¹ is H; R², R³, and R⁴ are independently selectedfrom the group consisting of H, optionally substituted C₁₋₁₈ alkyl,optionally substituted C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted C₆₋₁₀aryl-C₁₋₆ alkyl, optionally substituted 6- to 10-membered heteroaryl,and optionally substituted 6- to 10-membered heterocyclyl, provided thatat least one of R², R³, and R⁴ is other than H; R⁵ and R⁶ areindependently selected from the group consisting of H, optionallysubstituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈ haloalkyl,optionally substituted C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to10-membered heteroaryl, optionally substituted 6- to 10-memberedheterocyclyl, cyano, halo, N(R⁸)₂, B(R⁹)₂, Si(R⁹)₃, C(O)OR⁷, C(O)SR⁷,C(O)N(R⁷)₂, C(O)R⁷, C(O)ON(R⁷)₂, C(O)NR⁷OR⁸, C(O)C(O)OR⁷, andP(O)(OR⁷)₂; and each R⁷, R⁸, and R⁹ is independently selected from thegroup consisting of H and optionally substituted C₁₋₆ alkyl.
 15. Themethod of claim 14, wherein R² is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₆₋₁₀ aryl-C₁₋₆ alkyl, optionally substituted 6-to 10-membered heteroaryl, and optionally substituted 6- to 10-memberedheterocyclyl.
 16. The method of claim 14, wherein R³ and R⁴ are C₁₋₆alkyl.
 17. The method of claim 14, wherein R⁵ is C(O)OR⁷.
 18. The methodof claim 14, wherein R⁶ is selected from the group consisting ofoptionally substituted C₁₋₁₈ alkyl, optionally substituted C₁₋₁₈haloalkyl, and optionally substituted C₂₋₁₈ alkenyl.
 19. The method ofclaim 1, further comprising combining a reducing agent with thesilicon-containing reagent, the carbene precursor, and the heme protein.20. The method of claim 1, wherein the cytochrome c protein variant hasthe V75 mutation and the M100 mutation relative to the amino acidsequence set forth in SEQ ID NO:1.
 21. The method of claim 1, whereinthe cytochrome c protein variant has the M100 mutation and the M103mutation relative to the amino acid sequence set forth in SEQ ID NO:1.22. The method of claim 1, wherein the cytochrome c protein variant hasthe V75 mutation, the M100 mutation, and the M103 mutation relative tothe amino acid sequence set forth in SEQ ID NO:1.
 23. The method ofclaim 1, wherein the V75 mutation is a V75T mutation.
 24. The method ofclaim 1, wherein the M100 mutation is an M100D or M100E mutation. 25.The method of claim 1, wherein the M103 mutation is an M103E mutation.